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Sample records for patient dna samples

  1. High frequency of parvovirus B19 DNA in bone marrow samples from rheumatic patients

    DEFF Research Database (Denmark)

    Lundqvist, Anders; Isa, Adiba; Tolfvenstam, Thomas

    2005-01-01

    BACKGROUND: Human parvovirus B19 (B19) polymerase chain reaction (PCR) is now a routine analysis and serves as a diagnostic marker as well as a complement or alternative to B19 serology. The clinical significance of a positive B19 DNA finding is however dependent on the type of tissue or body fluid...... analysed and of the immune status of the patient. OBJECTIVES: To analyse the clinical significance of B19 DNA positivity in bone marrow samples from rheumatic patients. STUDY DESIGN: Parvovirus B19 DNA was analysed in paired bone marrow and serum samples by nested PCR technique. Serum was also analysed...... negative group. A high frequency of parvovirus B19 DNA was thus detected in bone marrow samples in rheumatic patients. The clinical data does not support a direct association between B19 PCR positivity and rheumatic disease manifestation. Therefore, the clinical significance of B19 DNA positivity in bone...

  2. Decreased mitochondrial DNA content in blood samples of patients with stage I breast cancer

    International Nuclear Information System (INIS)

    Xia, Peng; An, Han-Xiang; Dang, Cheng-Xue; Radpour, Ramin; Kohler, Corina; Fokas, Emmanouil; Engenhart-Cabillic, Rita; Holzgreve, Wolfgang; Zhong, Xiao Yan

    2009-01-01

    Alterations of mitochondrial DNA (mtDNA) have been implicated in carcinogenesis. We developed an accurate multiplex quantitative real-time PCR for synchronized determination of mtDNA and nuclear DNA (nDNA). We sought to investigate whether mtDNA content in the peripheral blood of breast cancer patients is associated with clinical and pathological parameters. Peripheral blood samples were collected from 60 patients with breast cancer and 51 age-matched healthy individuals as control. DNA was extracted from peripheral blood for the quantification of mtDNA and nDNA, using a one-step multiplex real-time PCR. A FAM labeled MGB probe and primers were used to amplify the mtDNA sequence of the ATP 8 gene, and a VIC labeled MGB probe and primers were employed to amplify the glyceraldehyde-3-phosphate-dehydrogenase gene. mtDNA content was correlated with tumor stage, menstruation status, and age of patients as well as lymph node status and the expression of estrogen receptor (ER), progesterone receptor (PR) and Her-2/neu protein. The content of mtDNA in stage I breast cancer patients was significantly lower than in other stages (overall P = 0.023). Reduced mtDNA was found often in post menopausal cancer group (P = 0.024). No difference in mtDNA content, in regards to age (p = 0.564), lymph node involvement (p = 0.673), ER (p = 0.877), PR (p = 0.763), and Her-2/neu expression (p = 0.335), was observed. Early detection of breast cancer has proved difficult and current detection methods are inadequate. In the present study, decreased mtDNA content in the peripheral blood of patients with breast cancer was strongly associated with stage I. The use of mtDNA may have diagnostic value and further studies are required to validate it as a potential biomarker for early detection of breast cancer

  3. Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients.

    Science.gov (United States)

    Ramírez, Juan Carlos; Cura, Carolina Inés; da Cruz Moreira, Otacilio; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Marcos da Matta Guedes, Paulo; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Maria da Cunha Galvão, Lúcia; Jácome da Câmara, Antonia Cláudia; Espinoza, Bertha; Alarcón de Noya, Belkisyole; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G

    2015-09-01

    An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. Public involvement in pharmacogenomics research: a national survey on patients' attitudes towards pharmacogenomics research and the willingness to donate DNA samples to a DNA bank in Japan.

    Science.gov (United States)

    Kobayashi, Eriko; Sakurada, Tomoya; Ueda, Shiro; Satoh, Nobunori

    2011-05-01

    To assess the attitude of Japanese patients towards pharmacogenomics research and a DNA bank for identifying genomic markers associated with adverse drug reactions (ADRs) and their willingness to donate DNA samples, we conducted a survey of 550 male and female patients. The majority of the respondents showed a positive attitude towards pharmacogenomics research (87.6%) and a DNA bank (75.1%). The willingness to donate DNA samples when experiencing severe ADRs (55.8%) was higher than when taking medications (40.4%). Positive attitudes towards a DNA bank and organ donation were significantly associated with an increased willingness to donate. Though the level of positive attitude in the patient population was higher than that in the general public in our former study (81.0 and 70.4%, respectively), the level of the willingness of patients to donate was 40.4% when taking medications and 55.8% when experiencing severe ADRs which was lower than that of the general public in our former study (45.3 and 61.7%). The results suggested that the level of true willingness in the patient population was lower than that of the general public considering the fictitious situation presented to the public (to suppose that they were patients receiving medication). It is important to assess the willingness of patients who are true potential donors, not the general public.

  5. Human herpes virus-8 DNA in bronchoalveolar lavage samples from patients with AIDS-associated pulmonary Kaposi's sarcoma

    DEFF Research Database (Denmark)

    Benfield, T L; Dodt, K K; Lundgren, Jens Dilling

    1997-01-01

    of KS. We hypothesized that these sequences are present in samples obtained by bronchoalveolar lavage (BAL) in patients with pulmonary KS. Utilizing a nested polymerase chain reaction (PCR), 7/12 BAL cell samples from HIV-infected patients with endobronchial KS were positive for HHV-8 DNA. In contrast......, and PCR amplification of HHV-8 in BAL cells provides a non-invasive method with a high positive predictive value....

  6. Quantitative detection of epstein-barr virus DNA in cerebrospinal fluid and blood samples of patients with relapsing-remitting multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Clementina E Cocuzza

    Full Text Available The presence of Epstein-Barr Virus (EBV DNA in cerebrospinal fluid (CSF and peripheral blood (PB samples collected from 55 patients with clinical and radiologically-active relapsing-remitting MS (RRMS and 51 subjects with other neurological diseases was determined using standardized commercially available kits for viral nucleic acid extraction and quantitative EBV DNA detection. Both cell-free and cell-associated CSF and PB fractions were analyzed, to distinguish latent from lytic EBV infection. EBV DNA was detected in 5.5% and 18.2% of cell-free and cell-associated CSF fractions of patients with RRMS as compared to 7.8% and 7.8% of controls; plasma and peripheral blood mononuclear cells (PBMC positivity rates were 7.3% and 47.3% versus 5.8% and 31.4%, respectively. No significant difference in median EBV viral loads of positive samples was found between RRMS and control patients in all tested samples. Absence of statistically significant differences in EBV positivity rates between RRMS and control patients, despite the use of highly sensitive standardized methods, points to the lack of association between EBV and MS disease activity.

  7. DNA Sampling Hook

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The DNA Sampling Hook is a significant improvement on a method of obtaining a tissue sample from a live fish in situ from an aquatic environment. A tissue sample...

  8. Authentication of forensic DNA samples.

    Science.gov (United States)

    Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon

    2010-02-01

    Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

  9. DNA biosensor for detection of Salmonella typhi from blood sample of typhoid fever patient using gold electrode modified by self-assembled monolayers of thiols

    Science.gov (United States)

    Suryapratiwi, Windha Novita; Paat, Vlagia Indira; Gaffar, Shabarni; Hartati, Yeni Wahyuni

    2017-05-01

    Electrochemical biosensors are currently being developed in order to handle various clinical problems in diagnosing infectious diseases caused by pathogenic bacteria, or viruses. On this research, voltammetric DNA biosensor using gold electrode modified by thiols with self-assembled monolayers had been developed to detect a certain sequence of Salmonella typhi DNA from blood sample of typhoid fever patient. Thiol groups of cysteamines (Cys) and aldehyde groups from glutaraldehydes (Glu) were used as a link to increase the performance of gold electrode in detecting guanine oxidation signal of hybridized S. typhi DNA and ssDNA probe. Standard calibration method was used to determine analytical parameters from the measurements. The result shown that, the detection of S. typhi DNA from blood sample of typhoid fever patient can be carried out by voltammetry using gold electrode modified by self-assembled monolayers of thiols. A characteristic oxidation potential of guanine using Au/Cys/Gluwas obtained at +0.17 until +0.20 V. Limit of detection and limit of quantification from this measurements were 1.91μg mL-1 and 6.35 μg mL-1. The concentration of complement DNA from sample was 6.96 μg mL-1.

  10. Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

    International Nuclear Information System (INIS)

    Cao Hongbin; Banh, Alice; Kwok, Shirley; Shi Xiaoli; Wu, Simon; Krakow, Trevor; Khong, Brian; Bavan, Brindha; Bala, Rajeev; Pinsky, Benjamin A.; Colevas, Dimitrios; Pourmand, Nader; Koong, Albert C.; Kong, Christina S.; Le, Quynh-Thu

    2012-01-01

    Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16 INK4a staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using 18 F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

  11. High-resolution DNA content analysis of microbiopsy samples in oral lichen planus.

    Science.gov (United States)

    Pentenero, M; Monticone, M; Marino, R; Aiello, C; Marchitto, G; Malacarne, D; Giaretti, W; Gandolfo, S; Castagnola, P

    2017-04-01

    DNA aneuploidy has been reported to be a predictor of poor prognosis in both premalignant and malignant lesions. In oral lichen planus (OLP), this hypothesis remains to be proved. This study aimed to determine the rate of occurrence of DNA aneuploidy in patients with OLP by high-resolution DNA flow cytometry. Patients with OLP were consecutively enrolled. Tissue samples were subdivided for formalin fixation and routine histological assessment and for immediate storage at -20°C for later DNA ploidy analysis, which was performed by DAPI staining of the extracted nuclei and excitation with a UV lamp. The DNA aneuploid sublines were characterized by the DNA Index. A DNA aneuploid status was observed in two of 77 patients with OLP (2.6%). When considering the clinical aspect of the OLP lesions, both DNA aneuploid cases had a reticular clinical aspect. DNA aneuploidy is an uncommon event in OLP and less frequent compared to other non-dysplastic and non-OLP oral potentially malignant disorders. The extremely low rate of DNA aneuploidy could represent an occasional finding or reflect the low rate of malignant transformation observed in patients with OLP even if the real prognostic value of DNA ploidy analysis in patients with OLP remains to be confirmed. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. International study to evaluate PCR methods for detection of Trypanosoma cruzi DNA in blood samples from Chagas disease patients.

    Directory of Open Access Journals (Sweden)

    Alejandro G Schijman

    Full Text Available BACKGROUND: A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. METHODOLOGY/FINDINGS: An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU I, IV and VI (set A, human blood spiked with parasite cells (set B and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C. Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA, 13 satellite DNA (Sat-DNA and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05-0.5 parasites/mL whereas specific kDNA tests detected 5.10(-3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood. The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3-94.4%, specificity of 85

  13. 28 CFR 28.12 - Collection of DNA samples.

    Science.gov (United States)

    2010-07-01

    ... Homeland Security, collecting DNA samples from: (1) Aliens lawfully in, or being processed for lawful... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection...

  14. Diagnosis of becker muscular dystrophy: Results of Re-analysis of DNA samples.

    Science.gov (United States)

    Straathof, Chiara S M; Van Heusden, Dave; Ippel, Pieternella F; Post, Jan G; Voermans, Nicol C; De Visser, Marianne; Brusse, Esther; Van Den Bergen, Janneke C; Van Der Kooi, Anneke J; Verschuuren, Jan J G M; Ginjaar, Hendrika B

    2016-01-01

    The phenotype of Becker muscular dystrophy (BMD) is highly variable, and the disease may be underdiagnosed. We searched for new mutations in the DMD gene in a cohort of previously undiagnosed patients who had been referred in the period 1985-1995. All requests for DNA analysis of the DMD gene in probands with suspected BMD were re-evaluated. If the phenotype was compatible with BMD, and no deletions or duplications were detected, DNA samples were screened for small mutations. In 79 of 185 referrals, no mutation was found. Analysis could be performed on 31 DNA samples. Seven different mutations, including 3 novel ones, were found. Long-term clinical follow-up is described. Refining DNA analysis in previously undiagnosed cases can identify mutations in the DMD gene and provide genetic diagnosis of BMD. A delayed diagnosis can still be valuable for the proband or the relatives of BMD patients. © 2015 Wiley Periodicals, Inc.

  15. Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

    Science.gov (United States)

    Turingan, Rosemary S; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F

    2016-01-01

    Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample

  16. Detection of Toxoplasma gondii DNA in sera samples of mice experimentally infected

    Directory of Open Access Journals (Sweden)

    H. Langoni

    2006-04-01

    Full Text Available Detection of Toxoplasma gondii (T. gondii DNA in blood can help to diagnose the disease in its acute phase; however, it must be considered that hemoglobin, present in blood, can inhibit polymerase activity, making impracticable the detection of DNA in samples. Mice were experimentally infected via oral route with ME49 and BTU2 strains cysts and RH strain tachyzoites; polymerase chain reaction was used to detect T. gondii DNA in mice sera 18, 24, 48, 96, and 192 hours post infection (PI. Toxoplama gondii DNA was detected in only one animal infected with BTU2 strain, genotype III (isolated from a dog with neurological signs 18 hours PI. The agent's DNA was not detected in any sample of the other experimental groups. New studies must be carried out to verify the technique sensitivity in researches on this agent's genetic material using sera samples of acute-phase toxoplasmosis patients, especially in cases of immunosuppression.

  17. Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

    Directory of Open Access Journals (Sweden)

    Karina Hatamoto Kawasato

    2013-02-01

    Full Text Available Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP, the internal transcribed spacer 16S-23S rRNA (ITS and the cell division (FtsZ of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%, FtsZ (17.4% and ITS (21.7%, respectively. After the second round six positive samples were identified by nested-HSP (26%, eight by nested-ITS (34.8% and 18 by nested-FtsZ (78.2%, corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001, enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%. In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

  18. Using long ssDNA polynucleotides to amplify STRs loci in degraded DNA samples

    Science.gov (United States)

    Pérez Santángelo, Agustín; Corti Bielsa, Rodrigo M.; Sala, Andrea; Ginart, Santiago; Corach, Daniel

    2017-01-01

    Obtaining informative short tandem repeat (STR) profiles from degraded DNA samples is a challenging task usually undermined by locus or allele dropouts and peak-high imbalances observed in capillary electrophoresis (CE) electropherograms, especially for those markers with large amplicon sizes. We hereby show that the current STR assays may be greatly improved for the detection of genetic markers in degraded DNA samples by using long single stranded DNA polynucleotides (ssDNA polynucleotides) as surrogates for PCR primers. These long primers allow a closer annealing to the repeat sequences, thereby reducing the length of the template required for the amplification in fragmented DNA samples, while at the same time rendering amplicons of larger sizes suitable for multiplex assays. We also demonstrate that the annealing of long ssDNA polynucleotides does not need to be fully complementary in the 5’ region of the primers, thus allowing for the design of practically any long primer sequence for developing new multiplex assays. Furthermore, genotyping of intact DNA samples could also benefit from utilizing long primers since their close annealing to the target STR sequences may overcome wrong profiling generated by insertions/deletions present between the STR region and the annealing site of the primers. Additionally, long ssDNA polynucleotides might be utilized in multiplex PCR assays for other types of degraded or fragmented DNA, e.g. circulating, cell-free DNA (ccfDNA). PMID:29099837

  19. SYBR green-based detection of Leishmania infantum DNA using peripheral blood samples.

    Science.gov (United States)

    Ghasemian, Mehrdad; Gharavi, Mohammad Javad; Akhlaghi, Lame; Mohebali, Mehdi; Meamar, Ahmad Reza; Aryan, Ehsan; Oormazdi, Hormozd; Ghayour, Zahra

    2016-03-01

    Parasitological methods for the diagnosis of visceral leishmaniasis (VL) require invasive sampling procedures. The aim of this study was to detect Leishmania infantum (L. infantum) DNA by real time-PCR method in peripheral blood of symptomatic VL patient and compared its performance with nested PCR, an established molecular method with very high diagnostic indices. 47 parasitologically confirmed VL patients diagnosed by direct agglutination test (DAT > 3200), bone marrow aspiration and presented characteristic clinical features (fever, hepatosplenomegaly, and anemia) and 40 controls (non-endemic healthy control-30, Malaria-2, Toxoplasma gondii-2, Mycobacterium tuberculosis-2, HBV-1, HCV-1, HSV-1 and CMV-1) were enrolled in this study. SYBR-green based real time-PCR and nested PCR was performed to amplify the Kinetoplast DNA minicircle gene using the DNA extracted from Buffy coat. From among 47 patients, 45 (95.7 %) were positive by both nested-PCR and real time-PCR. These results indicate that real time-PCR was not only as sensitive as a nested-PCR assay for detection of Leishmania kDNA in clinical sample, but also more rapid. The advantage of real time-PCR based methods over nested-PCR is simple to perform, more faster in which nested-PCR requires post-PCR processing and reducing contamination risk.

  20. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics.

    Science.gov (United States)

    van Ginkel, Joost H; van den Broek, Daan A; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M; Huibers, Manon M H

    2017-10-01

    In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for detecting rare mutational targets. In order to perform ddPCR on blood samples, a standardized procedure for processing and analyzing blood samples is necessary to facilitate implementation into clinical practice. Therefore, we assessed the technical sample workup procedure for ddPCR on blood plasma samples. Blood samples from healthy individuals, as well as lung cancer patients were analyzed. We compared different methods and protocols for sample collection, storage, centrifugation, isolation, and quantification. Cell-free DNA (cfDNA) concentrations of several wild-type targets and BRAF and EGFR-mutant ctDNA concentrations quantified by ddPCR were primary outcome measurements. Highest cfDNA concentrations were measured in blood collected in serum tubes. No significant differences in cfDNA concentrations were detected between various time points of up to 24 h until centrifugation. Highest cfDNA concentrations were detected after DNA isolation with the Quick cfDNA Serum & Plasma Kit, while plasma isolation using the QIAamp Circulating Nucleic Acid Kit yielded the most consistent results. DdPCR results on cfDNA are highly dependent on multiple factors during preanalytical sample workup, which need to be addressed during the development of this diagnostic tool for cancer diagnostics in the future. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  1. Increased levels of mitochondrial DNA copy number in patients with vitiligo.

    Science.gov (United States)

    Vaseghi, H; Houshmand, M; Jadali, Z

    2017-10-01

    Oxidative stress is known to be involved in the pathogenesis of autoimmune diseases such as vitiligo. Evidence suggests that the human mitochondrial DNA copy number (mtDNAcn) is vulnerable to damage mediated by oxidative stress. The purpose of this study was to examine and compare peripheral blood mtDNAcn and oxidative DNA damage byproducts (8-hydroxy-2-deoxyguanosine; 8-OHdG) in patients with vitiligo and healthy controls (HCs). The relative mtDNAcn and the oxidative damage (formation of 8-OHdG in mtDNA) of each sample were determined by real-time quantitative PCR. Blood samples were obtained from 56 patients with vitiligo and 46 HCs. The mean mtDNAcn and the degree of mtDNA damage were higher in patients with vitiligo than in HCs. These data suggest that increase in mtDNAcn and oxidative DNA damage may be involved in the pathogenesis of vitiligo. © 2017 British Association of Dermatologists.

  2. [DNA quantification of blood samples pre-treated with pyramidon].

    Science.gov (United States)

    Zhu, Chuan-Hong; Zheng, Dao-Li; Ni, Rao-Zhi; Wang, Hai-Sheng; Ning, Ping; Fang, Hui; Liu, Yan

    2014-06-01

    To study DNA quantification and STR typing of samples pre-treated with pyramidon. The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accordance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extraction is the best method for STR profiling and DNA extraction.

  3. [Quality of DNA from archival pathological samples of gallbladder cancer].

    Science.gov (United States)

    Roa, Iván; de Toro, Gonzalo; Sánchez, Tamara; Slater, Jeannie; Ziegler, Anne Marie; Game, Anakaren; Arellano, Leonardo; Schalper, Kurt; de Aretxabala, Xabier

    2013-12-01

    The quality of the archival samples stored at pathology services could be a limiting factor for molecular biology studies. To determine the quality of DNA extracted from gallbladder cancer samples at different institutions. One hundred ninety four samples coming from five medical centers in Chile, were analyzed. DNA extraction was quantified determining genomic DNA concentration. The integrity of DNA was determined by polymerase chain reaction amplification of different length fragments of a constitutive gene (β-globin products of 110, 268 and 501 base pairs). The mean DNA concentration obtained in 194 gallbladder cancer samples was 48 ± 43.1 ng/µl. In 22% of samples, no amplification was achieved despite obtaining a mean DNA concentration of 58.3 ng/ul. In 81, 67 and 22% of samples, a DNA amplification of at least 110, 268 or 501 base pairs was obtained, respectively. No differences in DNA concentration according to the source of the samples were demonstrated. However, there were marked differences in DNA integrity among participating centers. Samples from public hospitals were of lower quality than those from private clinics. Despite some limitations, in 80% of cases, the integrity of DNA in archival samples from pathology services in our country would allow the use of molecular biology techniques.

  4. Non-destructive sampling of ancient insect DNA

    DEFF Research Database (Denmark)

    Thomsen, Philip Francis; Elias, Scott; Gilbert, Tom

    2009-01-01

    BACKGROUND: A major challenge for ancient DNA (aDNA) studies on insect remains is that sampling procedures involve at least partial destruction of the specimens. A recent extraction protocol reveals the possibility of obtaining DNA from past insect remains without causing visual morphological...... of 77-204 base pairs (-bp) in size using species-specific and general insect primers. CONCLUSION/SIGNIFICANCE: The applied non-destructive DNA extraction method shows promising potential on insect museum specimens of historical age as far back as AD 1820, but less so on the ancient permafrost......-preserved insect fossil remains tested, where DNA was obtained from samples up to ca. 26,000 years old. The non-frozen sediment DNA approach appears to have great potential for recording the former presence of insect taxa not normally preserved as macrofossils and opens new frontiers in research on ancient...

  5. The importance of sound methodology in environmental DNA sampling

    Science.gov (United States)

    T. M. Wilcox; K. J. Carim; M. K. Young; K. S. McKelvey; T. W. Franklin; M. K. Schwartz

    2018-01-01

    Environmental DNA (eDNA) sampling - which enables inferences of species’ presence from genetic material in the environment - is a powerful tool for sampling rare fishes. Numerous studies have demonstrated that eDNA sampling generally provides greater probabilities of detection than traditional techniques (e.g., Thomsen et al. 2012; McKelvey et al. 2016; Valentini et al...

  6. Extraction of DNA from Forensic Biological Samples for Genotyping.

    Science.gov (United States)

    Stray, J E; Liu, J Y; Brevnov, M G; Shewale, J G

    2010-07-01

    Biological forensic samples constitute evidence with probative organic matter. Evidence believed to contain DNA is typically processed for extraction and purification of its nucleic acid content. Forensic DNA samples are composed of two things, a tissue and the substrate it resides on. Compositionally, a sample may contain almost anything and for each, the type, integrity, and content of both tissue and substrate will vary, as will the contaminant levels. This fact makes the success of extraction one of the most unpredictable steps in genotypic analysis. The development of robust genotyping systems and analysis platforms for short tandem repeat (STR) and mitochondrial DNA sequencing and the acceptance of results generated by these methods in the court system, resulted in a high demand for DNA testing. The increasing variety of sample submissions created a need to isolate DNA from forensic samples that may be compromised or contain low levels of biological material. In the past decade, several robust chemistries and isolation methods have been developed to safely and reliably recover DNA from a wide array of sample types in high yield and free of PCR inhibitors. In addition, high-throughput automated workflows have been developed to meet the demand for processing increasing numbers of samples. This review summarizes a number of the most widely adopted methods and the best practices for DNA isolation from forensic biological samples, including manual, semiautomated, and fully automated platforms. Copyright © 2010 Central Police University.

  7. Long-term frozen storage of urine samples: a trouble to get PCR results in Schistosoma spp. DNA detection?

    Science.gov (United States)

    Fernández-Soto, Pedro; Velasco Tirado, Virginia; Carranza Rodríguez, Cristina; Pérez-Arellano, José Luis; Muro, Antonio

    2013-01-01

    Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples' storage or conditions for handling and DNA preservation and extraction methods. We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patients urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template. Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.

  8. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA) from Patients with Non-Small Cell Lung Cancer (NSCLC)

    Science.gov (United States)

    Sherwood, James L.; Corcoran, Claire; Brown, Helen; Sharpe, Alan D.; Musilova, Milena; Kohlmann, Alexander

    2016-01-01

    Introduction Non-invasive mutation testing using circulating tumour DNA (ctDNA) is an attractive premise. This could enable patients without available tumour sample to access more treatment options. Materials & Methods Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits. Results 2 hr incubation time and double plasma centrifugation (2000 x g) reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA). Reduced “contamination” and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT) (Streck), after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield. Conclusion This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous. PMID:26918901

  9. Optimised Pre-Analytical Methods Improve KRAS Mutation Detection in Circulating Tumour DNA (ctDNA from Patients with Non-Small Cell Lung Cancer (NSCLC.

    Directory of Open Access Journals (Sweden)

    James L Sherwood

    Full Text Available Non-invasive mutation testing using circulating tumour DNA (ctDNA is an attractive premise. This could enable patients without available tumour sample to access more treatment options.Peripheral blood and matched tumours were analysed from 45 NSCLC patients. We investigated the impact of pre-analytical variables on DNA yield and/or KRAS mutation detection: sample collection tube type, incubation time, centrifugation steps, plasma input volume and DNA extraction kits.2 hr incubation time and double plasma centrifugation (2000 x g reduced overall DNA yield resulting in lowered levels of contaminating genomic DNA (gDNA. Reduced "contamination" and increased KRAS mutation detection was observed using cell-free DNA Blood Collection Tubes (cfDNA BCT (Streck, after 72 hrs following blood draw compared to EDTA tubes. Plasma input volume and use of different DNA extraction kits impacted DNA yield.This study demonstrated that successful ctDNA recovery for mutation detection in NSCLC is dependent on pre-analytical steps. Development of standardised methods for the detection of KRAS mutations from ctDNA specimens is recommended to minimise the impact of pre-analytical steps on mutation detection rates. Where rapid sample processing is not possible the use of cfDNA BCT tubes would be advantageous.

  10. A non-destructive DNA sampling technique for herbarium specimens.

    Science.gov (United States)

    Shepherd, Lara D

    2017-01-01

    Herbarium specimens are an important source of DNA for plant research but current sampling methods require the removal of material for DNA extraction. This is undesirable for irreplaceable specimens such as rare species or type material. Here I present the first non-destructive sampling method for extracting DNA from herbarium specimens. DNA was successfully retrieved from robust leaves and/or stems of herbarium specimens up to 73 years old.

  11. Quantification of damage in DNA recovered from highly degraded samples – a case study on DNA in faeces

    Directory of Open Access Journals (Sweden)

    Eveson J Paige

    2006-08-01

    Full Text Available Abstract Background Poorly preserved biological tissues have become an important source of DNA for a wide range of zoological studies. Measuring the quality of DNA obtained from these samples is often desired; however, there are no widely used techniques available for quantifying damage in highly degraded DNA samples. We present a general method that can be used to determine the frequency of polymerase blocking DNA damage in specific gene-regions in such samples. The approach uses quantitative PCR to measure the amount of DNA present at several fragment sizes within a sample. According to a model of random degradation the amount of available template will decline exponentially with increasing fragment size in damaged samples, and the frequency of DNA damage (λ can be estimated by determining the rate of decline. Results The method is illustrated through the analysis of DNA extracted from sea lion faecal samples. Faeces contain a complex mixture of DNA from several sources and different components are expected to be differentially degraded. We estimated the frequency of DNA damage in both predator and prey DNA within individual faecal samples. The distribution of fragment lengths for each target fit well with the assumption of a random degradation process and, in keeping with our expectations, the estimated frequency of damage was always less in predator DNA than in prey DNA within the same sample (mean λpredator = 0.0106 per nucleotide; mean λprey = 0.0176 per nucleotide. This study is the first to explicitly define the amount of template damage in any DNA extracted from faeces and the first to quantify the amount of predator and prey DNA present within individual faecal samples. Conclusion We present an approach for characterizing mixed, highly degraded PCR templates such as those often encountered in ecological studies using non-invasive samples as a source of DNA, wildlife forensics investigations and ancient DNA research. This method will

  12. Usefulness of FTA® cards as a Pneumocystis-DNA extraction method in bronchoalveolar lavage samples.

    Science.gov (United States)

    Rodiño, Jenniffer M; Aguilar, Yudy A; Rueda, Zulma Vanessa; Vélez, Lázaro A

    2016-01-01

    FTA® cards (Fast Technology for Analysis of Nucleic Acids) are an alternative DNA extraction method in bronchoalveolar lavage (BAL) samples for Pneumocystis jirovecii molecular analyses. The goal was to evaluate the usefulness of FTA® cards to detect P. jirovecii-DNA by PCR in BAL samples compared to silica adsorption chromatography (SAC). This study used 134 BAL samples from immunocompromised patients previously studied to establish microbiological aetiology of pneumonia, among them 15 cases of Pneumocystis pneumonia (PCP) documented by staining and 119 with other alternative diagnoses. The FTA® system and SAC were used for DNA extraction and then amplified by nested PCR to detect P. jirovecii. Performance and concordance of the two DNA extraction methods compared to P. jirovecii microscopy were calculated. The influence of the macroscopic characteristics, transportation of samples and the duration of the FTA® card storage (1, 7, 10 or 12 months) were also evaluated. Among 134 BAL samples, 56% were positive for P. jirovecii-DNA by SAC and 27% by FTA®. All 15 diagnosed by microscopy were detected by FTA® and SAC. Specificity of the FTA® system and SAC were 82.4% and 49.6%, respectively. Compared to SAC, positivity by FTA® decreased with the presence of blood in BAL (62% vs 13.5%). The agreement between samples at 7, 10 and 12 months was 92.5% for FTA®. Positive cases by FTA® remained the same after shipment by mail. Results suggest that FTA® is a practical, safe and economical method to preserve P. jirovecii-DNA in BAL samples for molecular studies.

  13. DNA Qualification Workflow for Next Generation Sequencing of Histopathological Samples

    Science.gov (United States)

    Simbolo, Michele; Gottardi, Marisa; Corbo, Vincenzo; Fassan, Matteo; Mafficini, Andrea; Malpeli, Giorgio; Lawlor, Rita T.; Scarpa, Aldo

    2013-01-01

    Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for

  14. DNA qualification workflow for next generation sequencing of histopathological samples.

    Directory of Open Access Journals (Sweden)

    Michele Simbolo

    Full Text Available Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF tissues, 6 formalin-fixed paraffin-embedded (FFPE tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard

  15. Hepatitis B virus DNA quantification with the three-in-one (3io) method allows accurate single-step differentiation of total HBV DNA and cccDNA in biopsy-size liver samples.

    Science.gov (United States)

    Taranta, Andrzej; Tien Sy, Bui; Zacher, Behrend Johan; Rogalska-Taranta, Magdalena; Manns, Michael Peter; Bock, Claus Thomas; Wursthorn, Karsten

    2014-08-01

    Hepatitis B virus (HBV) replicates via reverse transcription converting its partially double stranded genome into the covalently closed circular DNA (cccDNA). The long-lasting cccDNA serves as a replication intermediate in the nuclei of hepatocytes. It is an excellent, though evasive, parameter for monitoring the course of liver disease and treatment efficiency. To develop and test a new approach for HBV DNA quantification in serum and small-size liver samples. The p3io plasmid contains an HBV fragment and human β-actin gene (hACTB) as a standard. Respective TaqMan probes were labeled with different fluorescent dyes. A triplex real-time PCR for simultaneous quantification of total HBV DNA, cccDNA and hACTB could be established. Three-in-one method allows simultaneous analysis of 3 targets with a lower limit of quantification of 48 copies per 20 μl PCR reaction and a wide range of linearity (R(2)>0.99, pDNA samples from HBV infected patients. Total HBV DNA and cccDNA could be quantified in 32 and 22 of 33 FFPE preserved liver specimens, respectively. Total HBV DNA concentrations quantified by the 3io method remained comparable with Cobas TaqMan HBV Test v2.0. The three-in-one protocol allows the single step quantification of viral DNA in samples from different sources. Therefore lower sample input, faster data acquisition, a lowered error and significantly lower costs are the advantages of the method. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. [On the use of FTA technology for collection, archieving, and molecular analysis of microsporidia dna from clinical stool samples].

    Science.gov (United States)

    Sokolova, O I; Dem'ianov, A V; Bovers, L S; Did'e, E S; Sokolova, Iu Ia

    2011-01-01

    The FTA technology was applied for sampling, archiving, and molecular analysis of the DNA isolated from stool samples to diagnose and identify microsporidia, the intracellular opportunistic parasites which induce malabsortion syndrome in immunosuppressed humans, particularly in patients with AIDS. Microsporidia DNA was successfully amplified in 6 of 50 stool samples of HIV-positive patients of the S. P. Botkin Memorial Infectious Disease Hospital (St. Petersburg) applied to FTA cards (FTA-Cars, Whatman Inc. Florham Park, NJ, USA). Amplicons (the fragments of rDNA) were directly sequenced, and microsporidia species--Encephalitozoon intestinalis, E. cuniculi, E. hellem, and Enterocytozoon bieneusi--were identified in Genbank by NCBI BLAST program. The FTA method of DNA immobilization is especially promising for epidemiological and field population studies which involve genotyping of microsporidia species and isolates.

  17. Human herpesvirus-6 and -7 DNA in cerebrospinal fluid of facial palsy patients.

    Science.gov (United States)

    Kanerva, Mervi; Jääskeläinen, Anne J; Suvela, Minna; Piiparinen, Heli; Vaheri, Antti; Pitkäranta, Anne

    2008-04-01

    Finding human herpesvirus (HHV)-7 and dual HHV-6A and -6B DNA in cerebrospinal fluid (CSF) of two facial palsy (FP) patients is intriguing but does not allow etiologic conclusions as such. HHV-6 or -7 DNA was revealed in 10% of the CSF samples tested from 70 immunocompetent adolescents and adults; a highly unusual result. How these findings are associated with the diseases they accompany remains to be defined. To determine whether herpes simplex virus (HSV)-1 and -2, varicella-zoster virus (VZV), HHV-6A, -6B, and -7, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) DNA could be found in CSF of FP patients or controls. In all, 33 peripheral FP patients (26 idiopathic, 5 with herpesvirus infection, 1 puerperal, 1 Melkersson-Rosenthal syndrome) (34 CSF samples) and 36 controls (16 nonidiopathic FP, 7 hearing loss, 6 vertigo, 5 headache, 2 other) previously tested for HSV-1, VZV, and HHV-6 DNA by polymerase chain reaction (PCR) were tested with highly sensitive multiplex-PCR and an oligonucleotide microarray method. One FP patient had HHV-7 DNA and another had HHV-6A and -6B DNA simultaneously. In the control group, one HHV-7, one HHV-6A, and three HHV-6B DNA-positive specimens were found.

  18. Detection of Merkel Cell Polyomavirus DNA in Serum Samples of Healthy Blood Donors

    Science.gov (United States)

    Mazzoni, Elisa; Rotondo, John C.; Marracino, Luisa; Selvatici, Rita; Bononi, Ilaria; Torreggiani, Elena; Touzé, Antoine; Martini, Fernanda; Tognon, Mauro G.

    2017-01-01

    Merkel cell polyomavirus (MCPyV) has been detected in 80% of Merkel cell carcinomas (MCC). In the host, the MCPyV reservoir remains elusive. MCPyV DNA sequences were revealed in blood donor buffy coats. In this study, MCPyV DNA sequences were investigated in the sera (n = 190) of healthy blood donors. Two MCPyV DNA sequences, coding for the viral oncoprotein large T antigen (LT), were investigated using polymerase chain reaction (PCR) methods and DNA sequencing. Circulating MCPyV sequences were detected in sera with a prevalence of 2.6% (5/190), at low-DNA viral load, which is in the range of 1–4 and 1–5 copies/μl by real-time PCR and droplet digital PCR, respectively. DNA sequencing carried out in the five MCPyV-positive samples indicated that the two MCPyV LT sequences which were analyzed belong to the MKL-1 strain. Circulating MCPyV LT sequences are present in blood donor sera. MCPyV-positive samples from blood donors could represent a potential vehicle for MCPyV infection in receivers, whereas an increase in viral load may occur with multiple blood transfusions. In certain patient conditions, such as immune-depression/suppression, additional disease or old age, transfusion of MCPyV-positive samples could be an additional risk factor for MCC onset. PMID:29238698

  19. Long-term frozen storage of urine samples: a trouble to get PCR results in Schistosoma spp. DNA detection?

    Directory of Open Access Journals (Sweden)

    Pedro Fernández-Soto

    Full Text Available BACKGROUND: Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples' storage or conditions for handling and DNA preservation and extraction methods. METHODOLOGY/PRINCIPAL FINDINGS: We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patients urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template. CONCLUSIONS/SIGNIFICANCE: Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA

  20. Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

    Directory of Open Access Journals (Sweden)

    Elodie Caboux

    Full Text Available The European Prospective Investigation into Cancer and nutrition (EPIC is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88% performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.

  1. Microbial diversity in fecal samples depends on DNA extraction method

    DEFF Research Database (Denmark)

    Mirsepasi, Hengameh; Persson, Søren; Struve, Carsten

    2014-01-01

    was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). FINDINGS: In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I'Etoile, France......BACKGROUND: There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study...... by easyMag® from the same fecal samples. Furthermore, DNA extracts obtained using easyMag® seemed to contain inhibitory compounds, since in order to perform a successful PCR-analysis, the sample should be diluted at least 10 times. DGGE performed on PCR from DNA extracted by QIAamp DNA Stool Mini Kit DNA...

  2. Clinical Significance of Plasma Epstein-Barr Virus DNA in Pulmonary Lymphoepithelioma-like Carcinoma (LELC) Patients.

    Science.gov (United States)

    Xie, Mian; Wu, Xiaojun; Wang, Fang; Zhang, Jinjun; Ben, Xiaosong; Zhang, Jiexia; Li, Xiaoxiang

    2018-02-01

    Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is a histologically distinctive subtype of NSCLC and an Epstein-Barr virus (EBV)-associated epithelial neoplasm. We investigated the clinical significance of plasma concentrations of EBV DNA in patients with pulmonary LELC. Two independent sets of plasma samples from a total of 429 patients with patients with pulmonary LELC (287 initial and 142 confirmatory) were available for EBV DNA determination. Plasma samples from the patients were subjected to a real-time quantitative polymerase chain reaction before treatment and 3 months after radical resection. Cutoff points were determined for pretreatment plasma EBV DNA concentration (low disease status and change in EBV DNA concentrations by using nonparametric tests. High EBV DNA concentration was associated with shorter OS in the initial, confirmatory, and combined data sets (combined data set hazard ratio = 3.67, 95% confidence interval: 2.72-4.38, p disease. High EBV DNA concentration was also associated with shorter disease-free survival (DFS) in patients with stage I/II disease. Patients with persistently detectable plasma EBV DNA had significantly poorer OS (p disease progression of pulmonary LELC. High baseline EBV DNA concentration is an independent poor prognostic marker in patients with pulmonary LELC. These results should be confirmed in larger prospective trials. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

  3. Preanalytical blood sample workup for cell-free DNA analysis using Droplet Digital PCR for future molecular cancer diagnostics

    NARCIS (Netherlands)

    van Ginkel, Joost H.; van den Broek, Daan A.; van Kuik, Joyce; Linders, Dorothé; de Weger, Roel; Willems, Stefan M.; Huibers, Manon M.H.

    2017-01-01

    In current molecular cancer diagnostics, using blood samples of cancer patients for the detection of genetic alterations in plasma (cell-free) circulating tumor DNA (ctDNA) is an emerging practice. Since ctDNA levels in blood are low, highly sensitive Droplet Digital PCR (ddPCR) can be used for

  4. Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

    International Nuclear Information System (INIS)

    Liang Liang; Dou Peng; Dong Mingming; Ke Xiaokang; Bian Ningsheng; Liu Zhen

    2009-01-01

    Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 ± 0.83 x 10 -3 s -1 . The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

  5. The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients.

    Science.gov (United States)

    Ponti, Giovanni; Maccaferri, Monia; Manfredini, Marco; Kaleci, Shaniko; Mandrioli, Mauro; Pellacani, Giovanni; Ozben, Tomris; Depenni, Roberta; Bianchi, Giampaolo; Pirola, Giacomo Maria; Tomasi, Aldo

    2018-04-01

    Circulating cell-free tumor DNA (cfDNA) is of crucial interest in oncology. cfDNA constitutes a potential prognostic and therapeutic marker for different solid tumors and can be used in the diagnostic and therapeutic management of cancer patients for which nowadays there are no valid laboratory markers. In the present study, the quality and quantity of the cfDNA were assessed by different quantification procedures, in order to identify the potential applications of these techniques in the preliminary cfDNA quantification. Qubit with single (ss) and double strand (ds) DNA assay kits, NanoDrop and quantitative Real Time PCR (qPCR), were adopted to assess the cfDNA in the blood samples of 18 melanoma patients, 67 prostate cancer patients and 15 healthy controls. The quantification by NanoDrop (average value 8.48ng/μl, 95% confidence limit (CL)=7.23-9.73), Qubit ssDNA (average value 23.08ng/μl, CL=19.88-26.28), dsDNA (average value 4.32ng/μl, CL=3.52-5.12) assay kits and qPCR (average value 0.39ng/μl, CL=0.31-0.47) revealed differences among the four procedures. Qubit 2.0 ss-DNA kit gave higher cfDNA concentration values for all the samples analyzed. In detail, Qubit ssDNA assay revealed higher sensitivity in the quantification of small amounts of pure ss-DNA and ds-DNA, while NanoDrop allowed the assessment of the purity of cfDNA samples. The NanoDrop and Qubit 2.0 measurements were analyzed in order to define their correlation with qPCR cfDNA assessment, showing good correlation values with the qPCR that should be considered the "gold standard". In our proposal, the sequential combination of NanoDrop and Qubit ssDNA methods should be adopted for a cost-effective preliminary assessment of total circulating cfDNA in melanoma and prostate cancer patients, and only discordant values should undergo qPCR assessment. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Current developments in forensic interpretation of mixed DNA samples (Review)

    Science.gov (United States)

    HU, NA; CONG, BIN; LI, SHUJIN; MA, CHUNLING; FU, LIHONG; ZHANG, XIAOJING

    2014-01-01

    A number of recent improvements have provided contemporary forensic investigations with a variety of tools to improve the analysis of mixed DNA samples in criminal investigations, producing notable improvements in the analysis of complex trace samples in cases of sexual assult and homicide. Mixed DNA contains DNA from two or more contributors, compounding DNA analysis by combining DNA from one or more major contributors with small amounts of DNA from potentially numerous minor contributors. These samples are characterized by a high probability of drop-out or drop-in combined with elevated stutter, significantly increasing analysis complexity. At some loci, minor contributor alleles may be completely obscured due to amplification bias or over-amplification, creating the illusion of additional contributors. Thus, estimating the number of contributors and separating contributor genotypes at a given locus is significantly more difficult in mixed DNA samples, requiring the application of specialized protocols that have only recently been widely commercialized and standardized. Over the last decade, the accuracy and repeatability of mixed DNA analyses available to conventional forensic laboratories has greatly advanced in terms of laboratory technology, mathematical models and biostatistical software, generating more accurate, rapid and readily available data for legal proceedings and criminal cases. PMID:24748965

  7. Specific amplification of bacterial DNA by optimized so-called universal bacterial primers in samples rich of plant DNA.

    Science.gov (United States)

    Dorn-In, Samart; Bassitta, Rupert; Schwaiger, Karin; Bauer, Johann; Hölzel, Christina S

    2015-06-01

    Universal primers targeting the bacterial 16S-rRNA-gene allow quantification of the total bacterial load in variable sample types by qPCR. However, many universal primer pairs also amplify DNA of plants or even of archaea and other eukaryotic cells. By using these primers, the total bacterial load might be misevaluated, whenever samples contain high amounts of non-target DNA. Thus, this study aimed to provide primer pairs which are suitable for quantification and identification of bacterial DNA in samples such as feed, spices and sample material from digesters. For 42 primers, mismatches to the sequence of chloroplasts and mitochondria of plants were evaluated. Six primer pairs were further analyzed with regard to the question whether they anneal to DNA of archaea, animal tissue and fungi. Subsequently they were tested with sample matrix such as plants, feed, feces, soil and environmental samples. To this purpose, the target DNA in the samples was quantified by qPCR. The PCR products of plant and feed samples were further processed for the Single Strand Conformation Polymorphism method followed by sequence analysis. The sequencing results revealed that primer pair 335F/769R amplified only bacterial DNA in samples such as plants and animal feed, in which the DNA of plants prevailed. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. An Optimized DNA Analysis Workflow for the Sampling, Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints.

    Science.gov (United States)

    Solomon, April D; Hytinen, Madison E; McClain, Aryn M; Miller, Marilyn T; Dawson Cruz, Tracey

    2018-01-01

    DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints-touch DNA "sandwiched" between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post-amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri-Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7-100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases. © 2017 American Academy of Forensic Sciences.

  9. Study of microtip-based extraction and purification of DNA from human samples for portable devices

    Science.gov (United States)

    Fotouhi, Gareth

    DNA sample preparation is essential for genetic analysis. However, rapid and easy-to-use methods are a major challenge to obtaining genetic information. Furthermore, DNA sample preparation technology must follow the growing need for point-of-care (POC) diagnostics. The current use of centrifuges, large robots, and laboratory-intensive protocols has to be minimized to meet the global challenge of limited access healthcare by bringing the lab to patients through POC devices. To address these challenges, a novel extraction method of genomic DNA from human samples is presented by using heat-cured polyethyleneimine-coated microtips generating a high electric field. The microtip extraction method is based on recent work using an electric field and capillary action integrated into an automated device. The main challenges to the method are: (1) to obtain a stable microtip surface for the controlled capture and release of DNA and (2) to improve the recovery of DNA from samples with a high concentration of inhibitors, such as human samples. The present study addresses these challenges by investigating the heat curing of polyethyleneimine (PEI) coated on the surface of the microtip. Heat-cured PEI-coated microtips are shown to control the capture and release of DNA. Protocols are developed for the extraction and purification of DNA from human samples. Heat-cured PEI-coated microtip methods of DNA sample preparation are used to extract genomic DNA from human samples. It is discovered through experiment that heat curing of a PEI layer on a gold-coated surface below 150°C could inhibit the signal of polymerase chain reaction (PCR). Below 150°C, the PEI layer is not completely cured and dissolved off the gold-coated surface. Dissolved PEI binds with DNA to inhibit PCR. Heat curing of a PEI layer above 150°C on a gold-coated surface prevents inhibition to PCR and gel electrophoresis. In comparison to gold-coated microtips, the 225°C-cured PEI-coated microtips improve the

  10. DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fortes, F.P. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Kuasne, H. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Marchi, F.A. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Programa Inter-Institucional em Bioinformtica, Instituto de Matemtica e Estatstica, Universidade So Paulo, So Paulo, SP (Brazil); Miranda, P.M. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Rogatto, S.R. [CIPE, Laboratrio NeoGene, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Urologia, Faculdade de Medicina, Universidade Estadual Paulista, Botucatu, SP (Brazil); Achatz, M.I. [CIPE, Laboratrio de Oncogentica Molecular, A.C. Camargo Cancer Center, São Paulo, SP (Brazil); Departamento de Oncogentica, A.C. Camargo Cancer Center, So Paulo, SP (Brazil)

    2015-04-28

    Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

  11. 4977-bp mitochondrial DNA deletion in infertile patients with varicocele.

    Science.gov (United States)

    Gashti, N G; Salehi, Z; Madani, A H; Dalivandan, S T

    2014-04-01

    Varicocele is the abnormal inflexion and distension of veins of the pampiniform plexus within spermatic cord and is one of the amendable causes of male infertility. It can increase reactive oxygen species (ROS) production in semen and cause oxidative stress. The purpose of this study was to analyse spermatozoa mtDNA 4977-bp deletion in infertile men with varicocele. To detect 4977-bp deletion in spermatozoa mtDNA, semen samples of 60 infertile patients with clinical varicocele and 90 normal men from northern Iran were prepared. After extraction of spermatozoa total DNA, Gap polymerase chain reaction (Gap PCR) was performed. 4977-bp deletion was observed in 81.66% of patients with varicocele, while approximately 15.55% of controls had this deletion. As spermatozoa from patients with varicocele had a high frequency of occurrence of 4977-bp deletion in mtDNA [OR = 24.18, 95% confidence interval (CI) = 10.15-57.57, P deletion in spermatozoa and cause infertility in north Iranian men. However, to determine the relation between sperm mtDNA 4977-bp deletion and varicocele-induced infertility, larger population-based studies are needed. It is concluded that there is an association between sperm mtDNA 4977-bp deletion and varicocele-induced infertility in the population studied. © 2013 Blackwell Verlag GmbH.

  12. Development of a digital droplet PCR assay to measure HBV DNA in patients receiving long-term TDF treatment.

    Science.gov (United States)

    Liu, Yang; Cathcart, Andrea L; Delaney, William E; Kitrinos, Kathryn M

    2017-11-01

    The COBAS TaqMan assay has a lower limit of quantification (LLOQ) of 169 HBV copies/mL and a lower limit of detection (LLOD) of 58 copies/mL. HBV DNA below the TaqMan LLOQ is classified as target not detected (TND) (HBV DNA to 8 copies/mL. HBV DNA levels in plasma from patients with or without HBsAg seroconversion were assessed by ddPCR. For patients who did not achieve HBsAg seroconversion, the majority of TD samples (33/58, 57%) were HBV DNA positive by ddPCR while (10/37, 27%) of TND samples were positive. In contrast, for patients who achieved HBsAg seroconversion, HBV DNA was rarely detected by ddPCR after HBsAg seroconversion (1/28, 3.6%). ddPCR is a sensitive method to evaluate low-level viral replication in plasma samples. Frequent detection of HBV DNA by ddPCR among patients who did not achieve HBsAg seroconversion suggests new agents may be needed to suppress low levels of replicating HBV. Copyright © 2017. Published by Elsevier B.V.

  13. Quantitative analysis of DNA methylation in chronic lymphocytic leukemia patients.

    Science.gov (United States)

    Lyko, Frank; Stach, Dirk; Brenner, Axel; Stilgenbauer, Stephan; Döhner, Hartmut; Wirtz, Michaela; Wiessler, Manfred; Schmitz, Oliver J

    2004-06-01

    Changes in the genomic DNA methylation level have been found to be closely associated with tumorigenesis. In order to analyze the relation of aberrant DNA methylation to clinical and biological risk factors, we have determined the cytosine methylation level of 81 patients diagnosed with chronic lymphocytic leukemia (CLL). The analysis was based on DNA hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with BODIPY FL EDA. Derivatives were separated by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. We analyzed potential correlations between DNA methylation levels and numerous patient parameters, including clinical observations and biological data. As a result, we observed a significant correlation with the immunoglobulin variable heavy chain gene (VH) mutation status. This factor has been repeatedly proposed as a reliable prognostic marker for CLL, which suggests that the methylation level might be a valuable factor in determining the prognostic outcome of CLL. We are now in the process of refining our method to broaden its application potential. In this context, we show here that the oxidation of the fluorescence marker in the samples and the evaporation of methanol in the electrolytes can be prevented by a film of paraffin oil. In summary, our results thus establish capillary electrophoresis as a valuable tool for analyzing the DNA methylation status of clinical samples.

  14. Effect of sample storage time on detection of hybridization signals in Checkerboard DNA-DNA hybridization.

    Science.gov (United States)

    do Nascimento, Cássio; Muller, Katia; Sato, Sandra; Albuquerque Junior, Rubens Ferreira

    2012-04-01

    Long-term sample storage can affect the intensity of the hybridization signals provided by molecular diagnostic methods that use chemiluminescent detection. The aim of this study was to evaluate the effect of different storage times on the hybridization signals of 13 bacterial species detected by the Checkerboard DNA-DNA hybridization method using whole-genomic DNA probes. Ninety-six subgingival biofilm samples were collected from 36 healthy subjects, and the intensity of hybridization signals was evaluated at 4 different time periods: (1) immediately after collecting (n = 24) and (2) after storage at -20 °C for 6 months (n = 24), (3) for 12 months (n = 24), and (4) for 24 months (n = 24). The intensity of hybridization signals obtained from groups 1 and 2 were significantly higher than in the other groups (p  0.05). The Checkerboard DNA-DNA hybridization method was suitable to detect hybridization signals from all groups evaluated, and the intensity of signals decreased significantly after long periods of sample storage.

  15. Performance of 2 commercial real-time polymerase chain reaction assays for the detection of Aspergillus and Pneumocystis DNA in bronchoalveolar lavage fluid samples from critical care patients.

    Science.gov (United States)

    Orsi, Carlotta Francesca; Gennari, William; Venturelli, Claudia; La Regina, Annunziata; Pecorari, Monica; Righi, Elena; Machetti, Marco; Blasi, Elisabetta

    2012-06-01

    This article investigates the performance of 2 commercial real-time polymerase chain reaction (PCR) assays, MycAssay™ Aspergillus (Myc(Asp)Assay) and MycAssay™ Pneumocystis (Myc(PCP)Assay), on the ABI 7300 platform for the detection of Aspergillus (Asp) or Pneumocystis jirovecii (Pj) DNA in bronchoalveolar lavage (BAL) samples from 20 patients. Operationally, patients enrolled were clustered into 3 groups: invasive aspergillosis group (IA, 7 patients), Pj pneumonia group (PCP, 8 patients), and negative control group (5 patients). All the IA patients were Myc(Asp)Assay positive, whereas 12 non-IA patients returned negative PCR results. Furthermore, 7 of 8 PCP patients were Myc(PCP)Assay positive, while 9 non-PCP patients were PCR negative. In conclusion, these data provide an early indication of the effectiveness of both the Myc(Asp)Assay and Myc(PCP)Assay on the ABI 7300 platform for the detection of either Asp or Pj DNA in BAL from patients with deep fungal infections. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Preservation and rapid purification of DNA from decomposing human tissue samples.

    Science.gov (United States)

    Sorensen, Amy; Rahman, Elizabeth; Canela, Cassandra; Gangitano, David; Hughes-Stamm, Sheree

    2016-11-01

    One of the key features to be considered in a mass disaster is victim identification. However, the recovery and identification of human remains are sometimes complicated by harsh environmental conditions, limited facilities, loss of electricity and lack of refrigeration. If human remains cannot be collected, stored, or identified immediately, bodies decompose and DNA degrades making genotyping more difficult and ultimately decreasing DNA profiling success. In order to prevent further DNA damage and degradation after collection, tissue preservatives may be used. The goal of this study was to evaluate three customized (modified TENT, DESS, LST) and two commercial DNA preservatives (RNAlater and DNAgard ® ) on fresh and decomposed human skin and muscle samples stored in hot (35°C) and humid (60-70% relative humidity) conditions for up to three months. Skin and muscle samples were harvested from the thigh of three human cadavers placed outdoors for up to two weeks. In addition, the possibility of purifying DNA directly from the preservative solutions ("free DNA") was investigated in order to eliminate lengthy tissue digestion processes and increase throughput. The efficiency of each preservative was evaluated based on the quantity of DNA recovered from both the "free DNA" in solution and the tissue sample itself in conjunction with the quality and completeness of downstream STR profiles. As expected, DNA quantity and STR success decreased with time of decomposition. However, a marked decrease in DNA quantity and STR quality was observed in all samples after the bodies entered the bloat stage (approximately six days of decomposition in this study). Similar amounts of DNA were retrieved from skin and muscle samples over time, but slightly more complete STR profiles were obtained from muscle tissue. Although higher amounts of DNA were recovered from tissue samples than from the surrounding preservative, the average number of reportable alleles from the "free DNA" was

  17. Serum Epstein-Barr virus DNA, detected by droplet digital PCR, correlates with disease activity in patients with rheumatoid arthritis.

    Science.gov (United States)

    Kuusela, Elina; Kouri, Vesa-Petteri; Olkkonen, Juri; Koivuniemi, Riitta; Äyräväinen, Leena; Rajamäki, Kristiina; Valleala, Heikki; Nordström, Dan; Leirisalo-Repo, Marjatta; Ainola, Mari; Eklund, Kari K

    2018-03-20

    To study the prevalence of asymptomatic activation of Epstein-Barr virus (EBV) in patients with rheumatoid arthritis (RA) and to analyse the correlation of serum EBV DNA with the disease activity. The level of EBV DNA was determined by droplet digital PCR assay from the serum of 46 DMARD naive early RA (ERA) and 22 chronic RA (CRA)-patients at study onset. Follow-up samples from 31 ERA and 16 CRA patients were obtained after starting or modifying the anti-rheumatic treatment. EBV DNA was also measured from 33 healthy controls and 9 patients with adult onset Still's disease (AOSD). Disease activity was assessed by the disease activity score (DAS28). At baseline, EBV DNA was detected in the serum of 7 of the 46 ERA patients all of whom had moderate or high disease activity. In the follow-up samples, 11 of 31 patients were EBV DNA positive. At baseline EBV positive patients had significantly higher disease activity (p=0.036) and the concentration of EBV DNA correlated significantly with DAS28 (rs=0.333, p=0.024). EBV DNA was detected in 3 of 22 CRA patients at study onset and in 8 of 16 in the follow-up samples. At follow-up EBV positive patients had significantly higher DAS28 (p=0.027) and the concentration of EBV DNA correlated significantly with DAS28 (rs=0.724, p=0.002). Only one of the healthy controls and none of the AOSD patients were positive for EBV DNA. Active RA is associated with a lytic EBV infection which may have a role in the pathogenesis of RA.

  18. DNA cards: determinants of DNA yield and quality in collecting genetic samples for pharmacogenetic studies.

    Science.gov (United States)

    Mas, Sergi; Crescenti, Anna; Gassó, Patricia; Vidal-Taboada, Jose M; Lafuente, Amalia

    2007-08-01

    As pharmacogenetic studies frequently require establishment of DNA banks containing large cohorts with multi-centric designs, inexpensive methods for collecting and storing high-quality DNA are needed. The aims of this study were two-fold: to compare the amount and quality of DNA obtained from two different DNA cards (IsoCode Cards or FTA Classic Cards, Whatman plc, Brentford, Middlesex, UK); and to evaluate the effects of time and storage temperature, as well as the influence of anticoagulant ethylenediaminetetraacetic acid on the DNA elution procedure. The samples were genotyped by several methods typically used in pharmacogenetic studies: multiplex PCR, PCR-restriction fragment length polymorphism, single nucleotide primer extension, and allelic discrimination assay. In addition, they were amplified by whole genome amplification to increase genomic DNA mass. Time, storage temperature and ethylenediaminetetraacetic acid had no significant effects on either DNA card. This study reveals the importance of drying blood spots prior to isolation to avoid haemoglobin interference. Moreover, our results demonstrate that re-isolation protocols could be applied to increase the amount of DNA recovered. The samples analysed were accurately genotyped with all the methods examined herein. In conclusion, our study shows that both DNA cards, IsoCode Cards and FTA Classic Cards, facilitate genetic and pharmacogenetic testing for routine clinical practice.

  19. Searching for the Optimal Sampling Solution: Variation in Invertebrate Communities, Sample Condition and DNA Quality.

    Directory of Open Access Journals (Sweden)

    Martin M Gossner

    Full Text Available There is a great demand for standardising biodiversity assessments in order to allow optimal comparison across research groups. For invertebrates, pitfall or flight-interception traps are commonly used, but sampling solution differs widely between studies, which could influence the communities collected and affect sample processing (morphological or genetic. We assessed arthropod communities with flight-interception traps using three commonly used sampling solutions across two forest types and two vertical strata. We first considered the effect of sampling solution and its interaction with forest type, vertical stratum, and position of sampling jar at the trap on sample condition and community composition. We found that samples collected in copper sulphate were more mouldy and fragmented relative to other solutions which might impair morphological identification, but condition depended on forest type, trap type and the position of the jar. Community composition, based on order-level identification, did not differ across sampling solutions and only varied with forest type and vertical stratum. Species richness and species-level community composition, however, differed greatly among sampling solutions. Renner solution was highly attractant for beetles and repellent for true bugs. Secondly, we tested whether sampling solution affects subsequent molecular analyses and found that DNA barcoding success was species-specific. Samples from copper sulphate produced the fewest successful DNA sequences for genetic identification, and since DNA yield or quality was not particularly reduced in these samples additional interactions between the solution and DNA must also be occurring. Our results show that the choice of sampling solution should be an important consideration in biodiversity studies. Due to the potential bias towards or against certain species by Ethanol-containing sampling solution we suggest ethylene glycol as a suitable sampling solution when

  20. Inspecting Targeted Deep Sequencing of Whole Genome Amplified DNA Versus Fresh DNA for Somatic Mutation Detection: A Genetic Study in Myelodysplastic Syndrome Patients.

    Science.gov (United States)

    Palomo, Laura; Fuster-Tormo, Francisco; Alvira, Daniel; Ademà, Vera; Armengol, María Pilar; Gómez-Marzo, Paula; de Haro, Nuri; Mallo, Mar; Xicoy, Blanca; Zamora, Lurdes; Solé, Francesc

    2017-08-01

    Whole genome amplification (WGA) has become an invaluable method for preserving limited samples of precious stock material and has been used during the past years as an alternative tool to increase the amount of DNA before library preparation for next-generation sequencing. Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by presenting somatic mutations in several myeloid-related genes. In this work, targeted deep sequencing has been performed on four paired fresh DNA and WGA DNA samples from bone marrow of MDS patients, to assess the feasibility of using WGA DNA for detecting somatic mutations. The results of this study highlighted that, in general, the sequencing and alignment statistics of fresh DNA and WGA DNA samples were similar. However, after variant calling and when considering variants detected at all frequencies, there was a high level of discordance between fresh DNA and WGA DNA (overall, a higher number of variants was detected in WGA DNA). After proper filtering, a total of three somatic mutations were detected in the cohort. All somatic mutations detected in fresh DNA were also identified in WGA DNA and validated by whole exome sequencing.

  1. Assessing genetic polymorphisms using DNA extracted from cells present in saliva samples

    Directory of Open Access Journals (Sweden)

    Nemoda Zsofia

    2011-12-01

    Full Text Available Abstract Background Technical advances following the Human Genome Project revealed that high-quality and -quantity DNA may be obtained from whole saliva samples. However, usability of previously collected samples and the effects of environmental conditions on the samples during collection have not been assessed in detail. In five studies we document the effects of sample volume, handling and storage conditions, type of collection device, and oral sampling location, on quantity, quality, and genetic assessment of DNA extracted from cells present in saliva. Methods Saliva samples were collected from ten adults in each study. Saliva volumes from .10-1.0 ml, different saliva collection devices, sampling locations in the mouth, room temperature storage, and multiple freeze-thaw cycles were tested. One representative single nucleotide polymorphism (SNP in the catechol-0-methyltransferase gene (COMT rs4680 and one representative variable number of tandem repeats (VNTR in the serotonin transporter gene (5-HTTLPR: serotonin transporter linked polymorphic region were selected for genetic analyses. Results The smallest tested whole saliva volume of .10 ml yielded, on average, 1.43 ± .77 μg DNA and gave accurate genotype calls in both genetic analyses. The usage of collection devices reduced the amount of DNA extracted from the saliva filtrates compared to the whole saliva sample, as 54-92% of the DNA was retained on the device. An "adhered cell" extraction enabled recovery of this DNA and provided good quality and quantity DNA. The DNA from both the saliva filtrates and the adhered cell recovery provided accurate genotype calls. The effects of storage at room temperature (up to 5 days, repeated freeze-thaw cycles (up to 6 cycles, and oral sampling location on DNA extraction and on genetic analysis from saliva were negligible. Conclusions Whole saliva samples with volumes of at least .10 ml were sufficient to extract good quality and quantity DNA. Using

  2. HBV-DNA in hemodialysis patients infected by HCV

    International Nuclear Information System (INIS)

    Arababadi, Mohammad Kazemi; Hassanshahi, Gholamhossein; Yousefi, Hassan

    2009-01-01

    End-stage renal disease patients on chronic hemodialysis (HD) patients are at risk for both hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, and they may coexist. To determine the prevalence and clinical impact of HBV and HCV infection, we studied poly chain reaction (PCR) and reverse transcription (RT)-PCR on the blood samples of 90 HD patients in Kerman, Iran. ELISA test was used to detect anti-HBc, anti-HBs and HBs Ag. We found that 30 out of 90 (33.3%) patients were PCR-RT-PCR positive for HCV-RNA. No HBV-DNA (0%) was detected through the PCR study in both positive and negative HCV-RNA patient groups. Though none of the samples was HBsAg positive, 10 (33.3%) HCV-RNA positive patients were anti-HBc positive, and 12 (40.7%) were anti-HBs positive. We conclude that prevalence of hepatitis C infection is high in HD patients in our region, but not associated with active HBV infection. (author)

  3. Public involvement in pharmacogenomics research: a national survey on public attitudes towards pharmacogenomics research and the willingness to donate DNA samples to a DNA bank in Japan.

    Science.gov (United States)

    Kobayashi, Eriko; Satoh, Nobunori

    2009-11-01

    To assess the attitudes of the Japanese general public towards pharmacogenomics research and a DNA bank for identifying genomic markers associated with ADRs and their willingness to donate DNA samples, we conducted a national survey for 1,103 Japanese adults from the general public, not a patient population. The response rate was 36.8%. The majority of the respondents showed a positive attitude towards pharmacogenomics research (81.0%) and a DNA bank (70.4%). Considering fictitious clinical situations such as taking medications and experiencing ADRs, the willingness to donate DNA samples when experiencing ADRs (61.7%) was higher than when taking medications (45.3%). Older generations were significantly associated with a decreased willingness to donate (OR = 0.45, CI 0.28-0.72 in 50s. OR = 0.49, CI: 0.31-0.77 in 60s). Positive attitudes towards pharmacogenomics research, a DNA bank, blood/bone marrow/organ donation were significantly associated with an increased willingness. However, the respondents had the following concerns regarding a DNA bank: the confidentiality of their personal information, the manner by which research results were utilized and simply the use of their own DNA for research. In order to attain public understanding to overcome these concerns, a process of public awareness should be put into place to emphasize the beneficial aspects of identifying genomic markers associated with ADRs and to address these concerns raised in our study. Further study is needed to assess the willingness of actual patients taking medications in real situations, since the respondents in our study were from the general public, not a patient population, and their willingness was assessed on the condition of assuming that they were patients taking medications.

  4. A 'feather-trap' for collecting DNA samples from birds.

    Science.gov (United States)

    Maurer, Golo; Beck, Nadeena; Double, Michael C

    2010-01-01

    Genetic analyses of birds are usually based on DNA extracted from a blood sample. For some species, however, obtaining blood samples is difficult because they are sensitive to handling, pose a conservation or animal welfare concern, or evade capture. In such cases, feathers obtained from live birds in the wild can provide an alternative source of DNA. Here, we provide the first description and evaluation of a 'feather-trap', consisting of small strips of double-sided adhesive tape placed close to a nest with chicks, as a simple, inexpensive and minimally invasive method to collect feathers. The feather-trap was tested in tropical conditions on the Australian pheasant coucal (Centropus phasianinus). None of the 12 pairs of coucals on which the feather-trap was used abandoned the nest, and feeding rates did not differ from those of birds not exposed to a feather-trap. On average, 4.2 feathers were collected per trap over 2-5 days and, despite exposure to monsoonal rain, DNA was extracted from 71.4% of samples, albeit at low concentrations. The amount of genomic DNA extracted from each feather was sufficient to reliably genotype individuals at up to five microsatellite loci for parentage analysis. We show that a feather-trap can provide a reliable alternative for obtaining DNA in species where taking blood is difficult. It may also prove useful for collecting feather samples for other purposes, e.g. stable-isotope analysis. © 2009 Blackwell Publishing Ltd.

  5. DNA methylation analysis from saliva samples for epidemiological studies.

    Science.gov (United States)

    Nishitani, Shota; Parets, Sasha E; Haas, Brian W; Smith, Alicia K

    2018-06-18

    Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.

  6. DNA damage in preserved specimens and tissue samples: a molecular assessment

    Directory of Open Access Journals (Sweden)

    Cantin Elizabeth

    2008-10-01

    Full Text Available Abstract The extraction of genetic information from preserved tissue samples or museum specimens is a fundamental component of many fields of research, including the Barcode of Life initiative, forensic investigations, biological studies using scat sample analysis, and cancer research utilizing formaldehyde-fixed, paraffin-embedded tissue. Efforts to obtain genetic information from these sources are often hampered by an inability to amplify the desired DNA as a consequence of DNA damage. Previous studies have described techniques for improved DNA extraction from such samples or focused on the effect of damaging agents – such as light, oxygen or formaldehyde – on free nucleotides. We present ongoing work to characterize lesions in DNA samples extracted from preserved specimens. The extracted DNA is digested to single nucleosides with a combination of DNase I, Snake Venom Phosphodiesterase, and Antarctic Phosphatase and then analyzed by HPLC-ESI-TOF-MS. We present data for moth specimens that were preserved dried and pinned with no additional preservative and for frog tissue samples that were preserved in either ethanol, or formaldehyde, or fixed in formaldehyde and then preserved in ethanol. These preservation methods represent the most common methods of preserving animal specimens in museum collections. We observe changes in the nucleoside content of these samples over time, especially a loss of deoxyguanosine. We characterize the fragmentation state of the DNA and aim to identify abundant nucleoside lesions. Finally, simple models are introduced to describe the DNA fragmentation based on nicks and double-strand breaks.

  7. Applications of pooled DNA samples to the assessment of population affinities: short tandem repeats.

    Science.gov (United States)

    Crawford, M H; Banerjee, P; Demarchi, D A; Zlojutro, M; McComb, J; Livshits, G; Henneberg, M; Mosher, M J; Schanfield, M S; Knowles, J A

    2005-12-01

    Pooled DNA samples have been used in association studies of Mendelian disease genes. This method involves combining equal quantities of DNA from patients and control subjects into separate pools and comparing the pools for distributions of genetic markers. In this study identical quantities of DNA from 300 individuals representing 6 populations were pooled and amplified for 296 loci using the touchdown polymerase chain reaction (PCR) method. The purpose of this study is to test the efficacy of pooled DNA markers in the reconstruction of the genetic structure of human populations. The populations sampled included Chuvash, Buryats, Kizhi, Native Americans, South Africans, and New York City whites. To test the accuracy of the allele-frequency distributions, we genotyped the Buryats and New York samples individually for six microsatellite markers and compared their frequencies to the allele frequencies derived from the electropherogram peak heights for the pooled DNA, producing a correlation of 0.9811 with a variance of less than 0.04. Two-dimensional scaling of genetic distances among the six populations produced clusters that reflected known historical relationships. A distance matrix was created using all 296 loci, and matrices based on individual chromosomes were correlated against the total matrix. As expected, the largest chromosomes had the highest correlations with the total matrix, whereas one of the smallest chromosomes, chromosome 22, had the lowest correlation and differed most from the combined STR distance matrix.

  8. Oxidatively-induced DNA damage and base excision repair in euthymic patients with bipolar disorder.

    Science.gov (United States)

    Ceylan, Deniz; Tuna, Gamze; Kirkali, Güldal; Tunca, Zeliha; Can, Güneş; Arat, Hidayet Ece; Kant, Melis; Dizdaroglu, Miral; Özerdem, Ayşegül

    2018-05-01

    Oxidatively-induced DNA damage has previously been associated with bipolar disorder. More recently, impairments in DNA repair mechanisms have also been reported. We aimed to investigate oxidatively-induced DNA lesions and expression of DNA glycosylases involved in base excision repair in euthymic patients with bipolar disorder compared to healthy individuals. DNA base lesions including both base and nucleoside modifications were measured using gas chromatography-tandem mass spectrometry and liquid chromatography-tandem mass spectrometry with isotope-dilution in DNA samples isolated from leukocytes of euthymic patients with bipolar disorder (n = 32) and healthy individuals (n = 51). The expression of DNA repair enzymes OGG1 and NEIL1 were measured using quantitative real-time polymerase chain reaction. The levels of malondialdehyde were measured using high performance liquid chromatography. Seven DNA base lesions in DNA of leukocytes of patients and healthy individuals were identified and quantified. Three of them had significantly elevated levels in bipolar patients when compared to healthy individuals. No elevation of lipid peroxidation marker malondialdehyde was observed. The level of OGG1 expression was significantly reduced in bipolar patients compared to healthy individuals, whereas the two groups exhibited similar levels of NEIL1 expression. Our results suggest that oxidatively-induced DNA damage occurs and base excision repair capacity may be decreased in bipolar patients when compared to healthy individuals. Measurement of oxidatively-induced DNA base lesions and the expression of DNA repair enzymes may be of great importance for large scale basic research and clinical studies of bipolar disorder. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Characterization of DNA antigens from immune complexes deposited in the skin of patients with systemic lupus erythematosus

    Institute of Scientific and Technical Information of China (English)

    曾凡钦; 尹若菲; 谭国珍; 郭庆; 许德清

    2004-01-01

    Background Skin lesions are common manifestations in systemic lupus erythematosus (SLE). It is still unknown what the definite pathogenesis of skin involvement was and whether DNA participated in it. Our study was designed to explore the pathogenetic role and nature of nuclear antigen (DNA) deposited in the skin lesions of patients with SLE.Methods Thirty skin samples from patients with SLE and 2 normal skin samples were studied. Extracellular DNA was evaluated by indirect immunofluorescence methods. The deposited immune complexes were extracted by cryoprecipitation, and DNA was then isolated with phenol and chloroform. DNA fragment sizes were detected by agarose gel electrophoresis. Finally, 8 different probes were used to analyze the origin of these DNA molecules using Dot hybridization.Results Extracellular DNA staining was found only in skin lesions, mainly those located in the basement membrane zone, vascular wall, and hair follicle wall. Normal skin and non-lesion SLE skin showed no fluorescence at locations outside the nuclei. There were no differences in the rate and intensity of extracellular DNA staining when comparing active phase to remission phase patients. No relationship was found between extracellular DNA and circulating anti-dsDNA antibodies. Deposited DNA fragments clustered into four bands of somewhat discrete sizes: 20 000 bp, 1300 bp, 800-900 bp, 100-200 bp. Small sized fragments (100-200 bp) were positively correlated with disease activity (P<0.05, r=0.407). Dot hybridization showed significant homology of the various extracellular DNA fragments examined with human genomic DNA, but not with DNA from the microorganisms and viruses we examined. There were also homologies between DNA samples from different individuals.Conclusions DNA and its immune complexes may contribute to the pathogenesis of skin lesions in SLE. These DNA molecules range in size from 100 bp to 20 kb and may be endogenous in origin.

  10. Near-Patient Sampling to Assist Infection Control—A Case Report and Discussion

    Science.gov (United States)

    Tang, Julian W.; Hoyle, Elizabeth; Moran, Sammy; Pareek, Manish

    2018-01-01

    Air sampling as an aid to infection control is still in an experimental stage, as there is no consensus about which air samplers and pathogen detection methods should be used, and what thresholds of specific pathogens in specific exposed populations (staff, patients, or visitors) constitutes a true clinical risk. This case report used a button sampler, worn or held by staff or left free-standing in a fixed location, for environmental sampling around a child who was chronically infected by a respiratory adenovirus, to determine whether there was any risk of secondary adenovirus infection to the staff managing the patient. Despite multiple air samples taken on difference days, coinciding with high levels of adenovirus detectable in the child’s nasopharyngeal aspirates (NPAs), none of the air samples contained any detectable adenovirus DNA using a clinically validated diagnostic polymerase chain reaction (PCR) assay. Although highly sensitive, in-house PCR assays have been developed to detect airborne pathogen RNA/DNA, it is still unclear what level of specific pathogen RNA/DNA constitutes a true clinical risk. In this case, the absence of detectable airborne adenovirus DNA using a conventional diagnostic assay removed the requirement for staff to wear surgical masks and face visors when they entered the child’s room. No subsequent staff infections or outbreaks of adenovirus have so far been identified. PMID:29385031

  11. Near-Patient Sampling to Assist Infection Control—A Case Report and Discussion

    Directory of Open Access Journals (Sweden)

    Julian W. Tang

    2018-01-01

    Full Text Available Air sampling as an aid to infection control is still in an experimental stage, as there is no consensus about which air samplers and pathogen detection methods should be used, and what thresholds of specific pathogens in specific exposed populations (staff, patients, or visitors constitutes a true clinical risk. This case report used a button sampler, worn or held by staff or left free-standing in a fixed location, for environmental sampling around a child who was chronically infected by a respiratory adenovirus, to determine whether there was any risk of secondary adenovirus infection to the staff managing the patient. Despite multiple air samples taken on difference days, coinciding with high levels of adenovirus detectable in the child’s nasopharyngeal aspirates (NPAs, none of the air samples contained any detectable adenovirus DNA using a clinically validated diagnostic polymerase chain reaction (PCR assay. Although highly sensitive, in-house PCR assays have been developed to detect airborne pathogen RNA/DNA, it is still unclear what level of specific pathogen RNA/DNA constitutes a true clinical risk. In this case, the absence of detectable airborne adenovirus DNA using a conventional diagnostic assay removed the requirement for staff to wear surgical masks and face visors when they entered the child’s room. No subsequent staff infections or outbreaks of adenovirus have so far been identified.

  12. Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples.

    Science.gov (United States)

    Børsting, Claus; Mogensen, Helle Smidt; Morling, Niels

    2013-05-01

    Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR amplification of tandem repeats. These artifacts may be avoided by typing bi-allelic markers instead of STRs. In this work, the SNPforID multiplex assay was used to type LtDNA. A sensitized SNP typing protocol was introduced, that increased signal strengths without increasing noise and without affecting the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two different consensus models were used to interpret the SNP data. Correct profiles with 42-49 SNPs were generated from the 50 and 100 pg experiments, whereas a few incorrect genotypes were included in the generated profiles from the 25 pg experiments. With the strict consensus model, between 35 and 48 SNPs were correctly typed in the 25 pg experiments and only one allele drop-out (error rate: 0.07%) was observed in the consensus profiles. A total of 28 crime case samples were selected for typing with the sensitized SNPforID protocol. The samples were previously typed with old STR kits during the crime case investigation and only partial profiles (0-6 STRs) were obtained. Eleven of the samples could not be quantified with the Quantifiler™ Human DNA Quantification kit because of partial or complete inhibition of the PCR. For eight of these samples, SNP typing was only possible when the buffer and DNA polymerase used in the original protocol was replaced with the AmpFℓSTR(®) SEfiler Plus™ Master Mix, which was developed specifically for challenging forensic samples. All

  13. Comparison of parasite loads in serum and blood samples from patients in acute and chronic phases of Chagas disease.

    Science.gov (United States)

    Hernández, Carolina; Teherán, Aníbal; Flórez, Carolina; Ramírez, Juan David

    2018-04-17

    Molecular methods have been developed for the detection and quantification of Trypanosoma cruzi DNA in blood samples from patients with Chagas disease. However, aspects of sample processing necessary for quantitative real-time PCR (qPCR), such as the addition of guanidine hydrochloride to whole blood samples, may limit timely access to molecular diagnosis. We analysed 169 samples from serum and guanidine-EDTA blood (GEB) obtained from patients in acute and chronic phases of Chagas disease. We applied qPCR targeted to the satellite DNA region. Finally, we compared the parasite loads and cycle of threshold values of the qPCR. The results confirmed the usefulness of serum samples for the detection and quantification of parasite DNA in patients with Chagas disease, especially in the acute phase. However, the parasite loads detected in serum samples from patients in the chronic phase were lower than those detected in GEB samples. The epidemiological implications of the findings are herein discussed.

  14. Human papillomavirus DNA detection in menstrual blood from patients with cervical intraepithelial neoplasia and condyloma acuminatum.

    Science.gov (United States)

    Wong, Sze Chuen Cesar; Au, Thomas Chi Chuen; Chan, Sammy Chung Sum; Chan, Charles Ming Lok; Lam, Money Yan Yee; Zee, Benny Chung Ying; Pong, Wei Mei; Chan, Anthony Tak Cheung

    2010-03-01

    The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases.

  15. Environmental DNA (eDNA sampling improves occurrence and detection estimates of invasive burmese pythons.

    Directory of Open Access Journals (Sweden)

    Margaret E Hunter

    Full Text Available Environmental DNA (eDNA methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR for the Burmese python (Python molurus bivittatus, Northern African python (P. sebae, boa constrictor (Boa constrictor, and the green (Eunectes murinus and yellow anaconda (E. notaeus. Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive

  16. Environmental DNA (eDNA) sampling improves occurrence and detection estimates of invasive burmese pythons.

    Science.gov (United States)

    Hunter, Margaret E; Oyler-McCance, Sara J; Dorazio, Robert M; Fike, Jennifer A; Smith, Brian J; Hunter, Charles T; Reed, Robert N; Hart, Kristen M

    2015-01-01

    Environmental DNA (eDNA) methods are used to detect DNA that is shed into the aquatic environment by cryptic or low density species. Applied in eDNA studies, occupancy models can be used to estimate occurrence and detection probabilities and thereby account for imperfect detection. However, occupancy terminology has been applied inconsistently in eDNA studies, and many have calculated occurrence probabilities while not considering the effects of imperfect detection. Low detection of invasive giant constrictors using visual surveys and traps has hampered the estimation of occupancy and detection estimates needed for population management in southern Florida, USA. Giant constrictor snakes pose a threat to native species and the ecological restoration of the Florida Everglades. To assist with detection, we developed species-specific eDNA assays using quantitative PCR (qPCR) for the Burmese python (Python molurus bivittatus), Northern African python (P. sebae), boa constrictor (Boa constrictor), and the green (Eunectes murinus) and yellow anaconda (E. notaeus). Burmese pythons, Northern African pythons, and boa constrictors are established and reproducing, while the green and yellow anaconda have the potential to become established. We validated the python and boa constrictor assays using laboratory trials and tested all species in 21 field locations distributed in eight southern Florida regions. Burmese python eDNA was detected in 37 of 63 field sampling events; however, the other species were not detected. Although eDNA was heterogeneously distributed in the environment, occupancy models were able to provide the first estimates of detection probabilities, which were greater than 91%. Burmese python eDNA was detected along the leading northern edge of the known population boundary. The development of informative detection tools and eDNA occupancy models can improve conservation efforts in southern Florida and support more extensive studies of invasive constrictors

  17. A combined method for DNA analysis and radiocarbon dating from a single sample.

    Science.gov (United States)

    Korlević, Petra; Talamo, Sahra; Meyer, Matthias

    2018-03-07

    Current protocols for ancient DNA and radiocarbon analysis of ancient bones and teeth call for multiple destructive samplings of a given specimen, thereby increasing the extent of undesirable damage to precious archaeological material. Here we present a method that makes it possible to obtain both ancient DNA sequences and radiocarbon dates from the same sample material. This is achieved by releasing DNA from the bone matrix through incubation with either EDTA or phosphate buffer prior to complete demineralization and collagen extraction utilizing the acid-base-acid-gelatinization and ultrafiltration procedure established in most radiocarbon dating laboratories. Using a set of 12 bones of different ages and preservation conditions we demonstrate that on average 89% of the DNA can be released from sample powder with minimal, or 38% without any, detectable collagen loss. We also detect no skews in radiocarbon dates compared to untreated samples. Given the different material demands for radiocarbon dating (500 mg of bone/dentine) and DNA analysis (10-100 mg), combined DNA and collagen extraction not only streamlines the sampling process but also drastically increases the amount of DNA that can be recovered from limited sample material.

  18. Droplet digital PCR for detection and quantification of circulating tumor DNA in plasma of head and neck cancer patients.

    Science.gov (United States)

    van Ginkel, Joost H; Huibers, Manon M H; van Es, Robert J J; de Bree, Remco; Willems, Stefan M

    2017-06-19

    During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR). TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data. In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2-422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667-156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%. Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.

  19. Investigation of epstein-barr virus and parvovirus b19 DNA in allogeneic stem cell transplant patients.

    Science.gov (United States)

    Atalay, Altay; Gökahmetoğlu, Selma; Durmaz, Süleyman; Kandemir, Idris; Sağlam, Derya; Kaynar, Leylagül; Eser, Bülent; Cetin, Mustafa; Kılıç, Hüseyin

    2014-06-01

    We aimed to investigate posttransplant Epstein-Barr virus (EBV) and parvovirus B19 DNA in allogeneic stem cell transplant patients between 2009 and 2010. Forty-five adult patients in whom allogeneic stem cell transplantation was performed between April 2009 and November 2010 in the Erciyes University Faculty of Medicine, Department of Internal Medicine, Division of Hematology and Oncology, were included in the study. EBV and parvovirus B19 DNA positivity was investigated by using real-time polymerase chain reaction technique in 135 plasma samples obtained after transplantation at between 1 and 6 months. Pretransplant serological markers of EBV and parvovirus B19 were provided from patient files. In 32 (71.1%) of the patients, EBV antibodies in the pretransplantation period were as follows: anti-EBNA-1 IgG (+), VCA IgM (-), and VCA IgG (+). In 2 patients (4.45%), these antibodies were as follows: anti-EBNA-1 IgG (+), VCA IgM (-), and VCA IgG (-). In 1 patient (2.2%), they were as follows: anti-EBNA-1 IgG (-), VCA IgM (-), and VCA IgG (+). EBV serological markers were negative in 2 (2.2%) out of 45 patients before transplantation. There was low DNA positivity (parvovirus B19 IgM was negative and IgG was positive, parvovirus B19 IgM was positive and IgG was negative in 1 (2.3%) patient. Parvovirus B19 DNA was not identified in any of the samples obtained from these 45 patients. In this study, EBV and parvovirus B19 DNA were investigated in allogeneic stem cell transplant patients. None of the patients developed PTLD and parvovirus B19 DNA positivity was not detected. However, this issue needs to be further evaluated in prospective, multicenter studies with larger series of patients.

  20. Identification of Forensic Samples via Mitochondrial DNA in the Undergraduate Biochemistry Laboratory

    Science.gov (United States)

    Millard, Julie T.; Pilon, André M.

    2003-04-01

    A recent forensic approach for identification of unknown biological samples is mitochondrial DNA (mtDNA) sequencing. We describe a laboratory exercise suitable for an undergraduate biochemistry course in which the polymerase chain reaction is used to amplify a 440 base pair hypervariable region of human mtDNA from a variety of "crime scene" samples (e.g., teeth, hair, nails, cigarettes, envelope flaps, toothbrushes, and chewing gum). Amplification is verified via agarose gel electrophoresis and then samples are subjected to cycle sequencing. Sequence alignments are made via the program CLUSTAL W, allowing students to compare samples and solve the "crime."

  1. Environmental DNA from seawater samples correlate with trawl catches of Subarctic, deepwater fishes

    DEFF Research Database (Denmark)

    Thomsen, Philip Francis; Møller, Peter Rask; Sigsgaard, Eva Egelyng

    2016-01-01

    such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope...... depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were...... found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites...

  2. Simple DNA extraction of urine samples: Effects of storage temperature and storage time.

    Science.gov (United States)

    Ng, Huey Hian; Ang, Hwee Chen; Hoe, See Ying; Lim, Mae-Lynn; Tai, Hua Eng; Soh, Richard Choon Hock; Syn, Christopher Kiu-Choong

    2018-06-01

    Urine samples are commonly analysed in cases with suspected illicit drug consumption. In events of alleged sample mishandling, urine sample source identification may be necessary. A simple DNA extraction procedure suitable for STR typing of urine samples was established on the Promega Maxwell ® 16 paramagnetic silica bead platform. A small sample volume of 1.7mL was used. Samples were stored at room temperature, 4°C and -20°C for 100days to investigate the influence of storage temperature and time on extracted DNA quantity and success rate of STR typing. Samples stored at room temperature exhibited a faster decline in DNA yield with time and lower typing success rates as compared to those at 4°C and -20°C. This trend can likely be attributed to DNA degradation. In conclusion, this study presents a quick and effective DNA extraction protocol from a small urine volume stored for up to 100days at 4°C and -20°C. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Human Papillomavirus DNA Detection in Menstrual Blood from Patients with Cervical Intraepithelial Neoplasia and Condyloma Acuminatum ▿

    Science.gov (United States)

    Wong, Sze Chuen Cesar; Au, Thomas Chi Chuen; Chan, Sammy Chung Sum; Chan, Charles Ming Lok; Lam, Money Yan Yee; Zee, Benny Chung Ying; Pong, Wei Mei; Chan, Anthony Tak Cheung

    2010-01-01

    The Papanicolaou test generates pain and embarrassment, and cytology screening has limited sensitivity for detection of cervical neoplasia. These factors urge the use of another screening test that can overcome these limitations. We explore a completely noninvasive method using detection of human papillomavirus (HPV) DNA in women's menstrual blood (MB). The participants were divided into 3 cohorts: (i) 235 patients with cervical intraepithelial neoplasia 3 (CIN 3) (n = 48), CIN 2 (n = 60), CIN 1 (n = 58), or condyloma acuminatum (CAC) (n = 69) before treatment or remission; (ii) from the first cohort of patients, 108 CIN 3 or CIN 2 patients after treatment and 62 CIN 1 or CAC patients after remission; and (iii) 323 apparently normal subjects (ANS) without any cervical disease. The HPV genotypes of the infected patients were confirmed by direct sequencing. Quantitative real-time PCR (QRT-PCR) was used to measure the MB HPV16 load for 15 infected patients. Results showed that the sensitivity, specificity, and positive and negative predictive values for detection of MB HPV DNA in samples from patients with CIN or CAC were 82.8%, 93.1%, 90.0%, and 87.9%, respectively. Moreover, MB HPV DNA was found in samples from 22.2% of CIN 3 or CIN 2 patients after treatment, 0.0% of CIN 1 or CAC patients after remission, and 8.1% of ANS, 4 of whom were found to have CIN 1 or CAC. Furthermore, QRT-PCR showed that the normalized MB HPV16 DNA copy numbers in samples from patients with CIN 1 to CIN 3 were significantly increased. These preliminary results suggested that MB HPV DNA is a potential noninvasive marker for these premalignant cervical diseases. PMID:20089764

  4. Computational analyses of ancient pathogen DNA from herbarium samples: challenges and prospects.

    Science.gov (United States)

    Yoshida, Kentaro; Sasaki, Eriko; Kamoun, Sophien

    2015-01-01

    The application of DNA sequencing technology to the study of ancient DNA has enabled the reconstruction of past epidemics from genomes of historically important plant-associated microbes. Recently, the genome sequences of the potato late blight pathogen Phytophthora infestans were analyzed from 19th century herbarium specimens. These herbarium samples originated from infected potatoes collected during and after the Irish potato famine. Herbaria have therefore great potential to help elucidate past epidemics of crops, date the emergence of pathogens, and inform about past pathogen population dynamics. DNA preservation in herbarium samples was unexpectedly good, raising the possibility of a whole new research area in plant and microbial genomics. However, the recovered DNA can be extremely fragmented resulting in specific challenges in reconstructing genome sequences. Here we review some of the challenges in computational analyses of ancient DNA from herbarium samples. We also applied the recently developed linkage method to haplotype reconstruction of diploid or polyploid genomes from fragmented ancient DNA.

  5. Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples.

    Science.gov (United States)

    Westen, Antoinette A; Matai, Anuska S; Laros, Jeroen F J; Meiland, Hugo C; Jasper, Mandy; de Leeuw, Wiljo J F; de Knijff, Peter; Sijen, Titia

    2009-09-01

    For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.

  6. The effect of swim-up and gradient sperm preparation techniques on deoxyribonucleic acid (DNA) fragmentation in subfertile patients.

    Science.gov (United States)

    Oguz, Yuksel; Guler, Ismail; Erdem, Ahmet; Mutlu, Mehmet Firat; Gumuslu, Seyhan; Oktem, Mesut; Bozkurt, Nuray; Erdem, Mehmet

    2018-03-23

    To compare the effect of two different sperm preparation techniques, including swim-up and gradient methods on sperm deoxyribonucleic acid (DNA) fragmentation status of semen samples from unexplained and mild male factor subfertile patients undergoing intrauterine insemination (IUI). A prospective randomized study was conducted in 65 subfertile patients, including 34 unexplained and 31 male factor infertility to compare basal and post-procedure DNA fragmentation rates in swim-up and gradient techniques. Sperm DNA fragmentation rates were evaluated by a sperm chromatin dispersion (SCD) test in two portions of each sample of semen that was prepared with either swim-up or gradient techniques. Sperm motility and morphology were also assessed based on WHO 2010 criteria. Swim-up but not gradient method yielded a statistically significant reduction in the DNA fragmented sperm rate after preparation as compared to basal rates, in the semen samples of both unexplained (41.85 ± 22.04 vs. 28.58 ± 21.93, p gradient) and mild male factor (46.61 ± 19.38 vs. 30.32 ± 18.20, p gradient) subgroups. Swim-up method significantly reduces sperm DNA fragmentation rates and may have some prognostic value on intrauterine insemination in patients with decreased sperm DNA integrity.

  7. Direct PCR amplification of forensic touch and other challenging DNA samples: A review.

    Science.gov (United States)

    Cavanaugh, Sarah E; Bathrick, Abigail S

    2018-01-01

    DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and quantification steps, which is not ideal for forensic evidence containing low levels of DNA. Direct PCR amplification of forensic unknown samples has been suggested as a means to circumvent extraction and quantification, thereby retaining the DNA typically lost during those procedures. Direct PCR amplification is a method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification, or quantification. It allows for maximum quantities of DNA to be targeted, minimizes opportunities for error and contamination, and reduces the time and monetary resources required to process samples, although data analysis may take longer as the increased DNA detection sensitivity of direct PCR may lead to more instances of complex mixtures. ISO 17025 accredited laboratories have successfully implemented direct PCR for limited purposes (e.g., high-throughput databanking analysis), and recent studies indicate that direct PCR can be an effective method for processing low-yield evidence samples. Despite its benefits, direct PCR has yet to be widely implemented across laboratories for the processing of evidentiary items. While forensic DNA laboratories are always interested in new methods that will maximize the quantity and quality of genetic information obtained from evidentiary items, there is often a lag between the advent of useful methodologies and their integration into laboratories. Delayed implementation of direct PCR of evidentiary items can be attributed to a variety of factors, including regulatory guidelines that prevent laboratories from omitting the quantification step when processing forensic unknown samples, as is the case in the United States, and, more broadly, a reluctance to validate a technique that is not widely used for evidence samples. The

  8. Preservation of RNA and DNA from mammal samples under field conditions.

    Science.gov (United States)

    Camacho-Sanchez, Miguel; Burraco, Pablo; Gomez-Mestre, Ivan; Leonard, Jennifer A

    2013-07-01

    Ecological and conservation genetics require sampling of organisms in the wild. Appropriate preservation of the collected samples, usually by cryostorage, is key to the quality of the genetic data obtained. Nevertheless, cryopreservation in the field to ensure RNA and DNA stability is not always possible. We compared several nucleic acid preservation solutions appropriate for field sampling and tested them on rat (Rattus rattus) blood, ear and tail tip, liver, brain and muscle. We compared the efficacy of a nucleic acid preservation (NAP) buffer for DNA preservation against 95% ethanol and Longmire buffer, and for RNA preservation against RNAlater (Qiagen) and Longmire buffer, under simulated field conditions. For DNA, the NAP buffer was slightly better than cryopreservation or 95% ethanol, but high molecular weight DNA was preserved in all conditions. The NAP buffer preserved RNA as well as RNAlater. Liver yielded the best RNA and DNA quantity and quality; thus, liver should be the tissue preferentially collected from euthanized animals. We also show that DNA persists in nonpreserved muscle tissue for at least 1 week at ambient temperature, although degradation is noticeable in a matter of hours. When cryopreservation is not possible, the NAP buffer is an economical alternative for RNA preservation at ambient temperature for at least 2 months and DNA preservation for at least 10 months. © 2013 John Wiley & Sons Ltd.

  9. Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study.

    Science.gov (United States)

    Albujja, Mohammed H; Bin Dukhyil, Abdul Aziz; Chaudhary, Abdul Rauf; Kassab, Ahmed Ch; Refaat, Ahmed M; Babu, Saranya Ramesh; Okla, Mohammad K; Kumar, Sachil

    2018-01-01

    An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/μL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable. © 2017 American Academy of Forensic Sciences.

  10. Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

    Directory of Open Access Journals (Sweden)

    Elena Ordoñez

    2013-01-01

    Full Text Available Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL, the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

  11. Mitochondrial and Nuclear DNA in Patients with Severe Polytrauma

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    M. Sh Khubutia

    2013-01-01

    Full Text Available The components of mitochondria from the cells damaged by injury are a key component for the development of systemic inflammatory response syndrome (SIRS under aseptic conditions. At the same time, there is a significant increase in the plasma level of mitochondrial DNA (mtDNA, which may be a prognostic marker for infectious complications in patients with severe polytrauma. Objective: to study the time course of changes in the serum levels of mtDNA and nuclear DNA (nDNA in healthy individuals and patients with polytrauma and to reveal its possible association with the development of infectious pulmonary complications and with mortality. Subjects and methods. Seven healthy volunteers and 25 polytrauma with polytrauma of a mean injury severity score (ISS of 40.2±9.2. Sixteen (64% patients developed purulent tracheobronchitis and pneumonia; 5 (20% patients died. The amount of mtDNA and nDNA was determined within the first at 12 and 24 hours, then on days 3 and 5—7 after injury by the authors’ modified procedure using as the exogenous control of a circular DNA molecule. The content of mtDNA and nDNA was expressed as absolute values, by taking the arithmetic mean values as 100% for the volunteers. Results. There was a more than 2.5-fold increase in mtDNA levels in dead patients as compared to survivors (p<0.05; the differences in nDMA levels were insignificant (p=0.1. Within the first 12 hours, the mean mtDNA level in patients with pneumonia was 34 times greater than the reference values and continued to rise in the following 12 hours whereas in those without pneumonia, it was only 17 times higher with its further decrease in the comparable time periods. In the first 12 hours, nDNA was increased in both groups, but 24 hours after injury it was 2555 times more than the reference value only in patients with pneumonia whereas it was decreased 3-fold in those without this condition. Conclusion. This paper is the first to describe the time course of

  12. The effects of storage temperature and duration of blood samples on DNA and RNA qualities.

    Science.gov (United States)

    Huang, Lien-Hung; Lin, Pei-Hsien; Tsai, Kuo-Wang; Wang, Liang-Jen; Huang, Ying-Hsien; Kuo, Ho-Chang; Li, Sung-Chou

    2017-01-01

    DNA and RNA samples from blood are the common examination target for non-invasive physical tests and/or biomedical studies. Since high-quality DNA and RNA samples guarantee the correctness of these tests and/or studies, we investigated the effects of storage temperature and storage duration of whole blood on DNA and RNA qualities. Subjects were enrolled to donate blood samples which were stored for different durations and at different temperatures, followed by the examinations on RNA quality, qPCR, DNA quality and DNA methylation. For RNA, we observed obvious quality decline with storage duration longer than 24 hours. Storage at low temperature does not keep RNA samples from degradation. And, storing whole blood samples in freezer dramatically damage RNA. For DNA, quality decline was not observed even with storage duration for 15 days. However, DNA methylation significantly altered with storage duration longer than three days. Storage duration within 24 hours is critical for collecting high-quality RNA samples for next-generation sequencing (NGS) assays (RIN≧8). If microarray assays are expected (RIN≧7), storage duration within 32 hours is acceptable. Although DNA is resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to WBC lysis. In addition, duration for more than three days significantly alter DNA methylation status, globally and locally. Our result provides a reference for dealing with blood samples.

  13. Novel and differential accumulation of mitochondrial DNA deletions in Swedish and vietnamese patients with colorectal cancer.

    Science.gov (United States)

    Dimberg, Jan; Hong, Thai Trinh; Skarstedt, Marita; Löfgren, Sture; Zar, Niklas; Matussek, Andreas

    2014-01-01

    Mitochondrial DNA (mtDNA) has been proposed to be involved in carcinogenesis and aging. The mtDNA 4977 bp deletion is one of the most frequently observed mtDNA mutations in human tissues and may play a role in colorectal cancer (CRC). In the present study, we aimed to evaluate the frequency of mtDNA 4977 bp deletion in CRC tissues and its association with clinical factors. We determined the presence of the 4977 bp common deletion in cancer and normal paired tissue samples from 105 Swedish and 88 Vietnamese patients with CRC using polymerase chain reaction (PCR) assays. The mtDNA 4977 bp deletion was shown to be significantly more frequent in normal tissues in comparison with paired cancer tissues in both Swedish and Vietnamese patients. The 4977 bp common deletion was significantly more frequent in cancer tissues of the Vietnamese patients compared to the Swedish patients, and in Vietnamese cancer tissues, the 4977 bp deletion was significantly over represented in those with localized disease compared to those with disseminated disease. Moreover, we detected nine novel mtDNA deletions and found a significantly higher rate of these in CRC tissues in Swedish in comparison to Vietnamese patients. The mtDNA 4977 bp deletion seems to have an impact on the clinical outcome of CRC in Vietnamese patients, that the Swedish patients accumulate more of the detected novel deletions in CRC tissue compared to Vietnamese patients probably indicates divergent mechanisms in colorectal carcinogenesis.

  14. Comparison of ESR1 Mutations in Tumor Tissue and Matched Plasma Samples from Metastatic Breast Cancer Patients

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    Takashi Takeshita

    2017-10-01

    Full Text Available BACKGROUND: ESR1 mutation in circulating cell-free DNA (cfDNA is emerging as a noninvasive biomarker of acquired resistance to endocrine therapy, but there is a paucity of data comparing the status of ESR1 gene in cfDNA with that in its corresponding tumor tissue. The objective of this study is to validate the degree of concordance of ESR1 mutations between plasma and tumor tissue. METHODS: ESR1 ligand-binding domain mutations Y537S, Y537N, Y537C, and D538G were analyzed using droplet digital PCR in 35 patients with metastatic breast cancer (MBC (35 tumor tissue samples and 67 plasma samples. RESULTS: Of the 35 paired samples, 26 (74.3% were concordant: one patient had detectable ESR1 mutations both plasma (ESR1 Y537S/Y537N and tumor tissue (ESR1 Y537S/Y537C, and 25 had WT ESR1 alleles in both. Nine (25.7% had discordance between the plasma and tissue results: five had mutations detected only in their tumor tissue (two Y537S, one Y537C, one D538G, and one Y537S/Y537N/D538G, and four had mutations detected only in their plasma (one Y537S, one Y537N, and two Y537S/Y537N/D538G. Furthermore, longitudinal plasma samples from 19 patients were used to assess changes in the presence of ESR1 mutations during treatment. Eleven patients had cfDNA ESR1 mutations over the course of treatment. A total of eight of 11 patients with MBC with cfDNA ESR1 mutations (72.7% had the polyclonal mutations. CONCLUSION: We have shown the independent distribution of ESR1 mutations between plasma and tumor tissue in 35 patients with MBC.

  15. Effect of DNA extraction and sample preservation method on rumen bacterial population.

    Science.gov (United States)

    Fliegerova, Katerina; Tapio, Ilma; Bonin, Aurelie; Mrazek, Jakub; Callegari, Maria Luisa; Bani, Paolo; Bayat, Alireza; Vilkki, Johanna; Kopečný, Jan; Shingfield, Kevin J; Boyer, Frederic; Coissac, Eric; Taberlet, Pierre; Wallace, R John

    2014-10-01

    The comparison of the bacterial profile of intracellular (iDNA) and extracellular DNA (eDNA) isolated from cow rumen content stored under different conditions was conducted. The influence of rumen fluid treatment (cheesecloth squeezed, centrifuged, filtered), storage temperature (RT, -80 °C) and cryoprotectants (PBS-glycerol, ethanol) on quality and quantity parameters of extracted DNA was evaluated by bacterial DGGE analysis, real-time PCR quantification and metabarcoding approach using high-throughput sequencing. Samples clustered according to the type of extracted DNA due to considerable differences between iDNA and eDNA bacterial profiles, while storage temperature and cryoprotectants additives had little effect on sample clustering. The numbers of Firmicutes and Bacteroidetes were lower (P rumen fluid subjected to the eDNA isolation procedure considerably changed the ratio of molecular operational taxonomic units (MOTUs) of Bacteroidetes and Firmicutes. Intracellular DNA extraction using bead-beating method from cheesecloth sieved rumen content mixed with PBS-glycerol and stored at -80 °C was found as the optimal method to study ruminal bacterial profile. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Ancient DNA and Forensics Mutual Benefits a Practical Sampling and Laboratory Guide Through a Virtual Ancient DNA Study

    Directory of Open Access Journals (Sweden)

    Jan Cemper-Kiesslich

    2014-09-01

    In this review the authors give a general overview on the field of ancient DNA analysis focussing of the potentials and limits, fields of application, requirements for samples, laboratory setup, reaction design and equipment as well as a brief outlook on current developments, future perspectives and potential cross links with associated scientific disciplines. Key words: Human DNA, Ancient DNA, Forensic DNA typing, Molecular archaeology, Application.

  17. Validated methodology for quantifying infestation levels of dreissenid mussels in environmental DNA (eDNA) samples.

    Science.gov (United States)

    Peñarrubia, Luis; Alcaraz, Carles; Vaate, Abraham Bij de; Sanz, Nuria; Pla, Carles; Vidal, Oriol; Viñas, Jordi

    2016-12-14

    The zebra mussel (Dreissena polymorpha Pallas, 1771) and the quagga mussel (D. rostriformis Deshayes, 1838) are successful invasive bivalves with substantial ecological and economic impacts in freshwater systems once they become established. Since their eradication is extremely difficult, their detection at an early stage is crucial to prevent spread. In this study, we optimized and validated a qPCR detection method based on the histone H2B gene to quantify combined infestation levels of zebra and quagga mussels in environmental DNA samples. Our results show specific dreissenid DNA present in filtered water samples for which microscopic diagnostic identification for larvae failed. Monitoring a large number of locations for invasive dreissenid species based on a highly specific environmental DNA qPCR assay may prove to be an essential tool for management and control plans focused on prevention of establishment of dreissenid mussels in new locations.

  18. Influence of Sampling Practices on the Appearance of DNA Image Histograms of Prostate Cells in FNAB Samples

    Directory of Open Access Journals (Sweden)

    Abdelbaset Buhmeida

    1999-01-01

    Full Text Available Twenty‐one fine needle aspiration biopsies (FNAB of the prostate, diagnostically classified as definitely malignant, were studied. The Papanicolaou or H&E stained samples were destained and then stained for DNA with the Feulgen reaction. DNA cytometry was applied after different sampling rules. The histograms varied according to the sampling rule applied. Because free cells between cell groups were easier to measure than cells in the cell groups, two sampling rules were tested in all samples: (i cells in the cell groups were measured, and (ii free cells between cell groups were measured. Abnormal histograms were more common after the sampling rule based on free cells, suggesting that abnormal patterns are best revealed through the free cells in these samples. The conclusions were independent of the applied histogram interpretation method.

  19. Epigenetic markers in circulating cell-free DNA as prognostic markers for survival of castration-resistant prostate cancer patients.

    Science.gov (United States)

    Hendriks, Rianne J; Dijkstra, Siebren; Smit, Frank P; Vandersmissen, Johan; Van de Voorde, Hendrik; Mulders, Peter F A; van Oort, Inge M; Van Criekinge, Wim; Schalken, Jack A

    2018-04-01

    Noninvasive biomarkers to guide personalized treatment for castration-resistant prostate cancer (CRPC) are needed. In this study, we analyzed hypermethylation patterns of two genes (GSTP1 and APC) in plasma cell-free DNA (cfDNA) of CRPC patients. The aim of this study was to analyze the cfDNA concentrations and levels of the epigenetic markers and to assess the value of these biomarkers for prognosis. In this prospective study, patients were included before starting new treatment after developing CRPC. The blood samples were collected prior to start of the treatment and at three time points thereafter. cfDNA was extracted from 1.5 mL of plasma and before performing a methylation-specific PCR, bisulfate modification was carried out. The median levels of cfDNA, GSTP1, and APC copies in the baseline samples of CRPC patients (n = 47) were higher than in controls (n = 30). In the survival analysis, the group with baseline marker levels below median had significant less PCa-related deaths (P-values Prostate Published by Wiley Periodicals, Inc.

  20. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types

    DEFF Research Database (Denmark)

    Lever, Mark; Torti, Andrea; Eickenbusch, Philip

    2015-01-01

    tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world...... DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample...

  1. Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

    Science.gov (United States)

    Zayas, Beatriz; Stillwell, Sara W; Wishnok, John S; Trudel, Laura J; Skipper, Paul; Yu, Mimi C; Tannenbaum, Steven R; Wogan, Gerald N

    2007-02-01

    We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

  2. Producing standard damaged DNA samples by heating: pitfalls and suggestions.

    Science.gov (United States)

    Fattorini, Paolo; Marrubini, Giorgio; Bonin, Serena; Bertoglio, Barbara; Grignani, Pierangela; Recchia, Elisa; Pitacco, Paola; Procopio, Francesca; Cantoni, Carolina; Pajnič, Irena Zupanič; Sorçaburu-Cigliero, Solange; Previderè, Carlo

    2018-05-15

    Heat-mediated hydrolysis of DNA is a simple and inexpensive method for producing damaged samples in vitro. Despite heat-mediated DNA hydrolysis is being widely used in forensic and clinical validation procedures, the lack of standardized procedures makes it impossible to compare the intra and inter-laboratory outcomes of the damaging treatments. In this work, a systematic approach to heat induced DNA hydrolysis was performed at 70 °C for 0-18 h to test the role both of the hydrolysis buffer and of the experimental conditions. Specifically, a trial DNA sample, resuspended in three different media (ultrapure water, 0.1% DEPC-water and, respectively, TE) was treated both in Eppendorf tubes ("Protocol P") and in Eppendorf tubes provided with screwcaps ("Protocol S"). The results of these comparative tests were assessed by normalization of the qPCR results. DEPC-water increased the degradation of the samples up to about 100 times when compared to the ultrapure water. Conversely, the TE protected the DNA from degradation whose level was about 1700 times lower than in samples treated in ultrapure water. Even the employment of the "Protocol S" affected the level of degradation, by consistently increasing it (up to about 180 times in DEPC-water). Thus, this comparative approach showed that even seemingly apparently trivial and often underestimated parameters modify the degradation level up to 2-3 orders of magnitude. The chemical-physical reasons of these findings are discussed together with the role of potential factors such as enhanced reactivity of CO 2 , ROS, NO x and pressure, which are likely to be involved. Since the intra and inter-laboratory comparison of the outcomes of the hydrolytic procedure is the first step toward its standardization, the normalization of the qPCR data by the UV/qPCR ratio seems to be the simplest and most reliable way to allow this. Finally, the supplying (provided with the commercial qPCR kits) of a DNA sample whose degree of

  3. Expression of DNA repair genes in ovarian cancer samples: biological and clinical considerations.

    Science.gov (United States)

    Ganzinelli, M; Mariani, P; Cattaneo, D; Fossati, R; Fruscio, R; Corso, S; Ricci, F; Broggini, M; Damia, G

    2011-05-01

    The purpose of this study was to investigate retrospectively the mRNA expression of genes involved in different DNA repair pathways implicated in processing platinum-induced damage in 171 chemotherapy-naïve ovarian tumours and correlate the expression of the different genes with clinical parameters. The expression of genes involved in DNA repair pathways (PARP1, ERCC1, XPA, XPF, XPG, BRCA1, FANCA, FANCC, FANCD2, FANCF and PolEta), and in DNA damage transduction (Chk1 and Claspin) was measured by RT-PCR in 13 stage I borderline and 77 stage I and 88 III ovarian carcinomas. ERCC1, XPA, XPF and XPG genes were significantly less expressed in stage III than in stage I carcinoma; BRCA1, FANCA, FANCC, FANCD2 gene expressions were low in borderline tumours, higher in stage I carcinomas and lower in stage III samples. High levels of ERCC1, XPA, FANCC, XPG and PolEta correlated with an increase in Overall Survival (OS) and Progression Free Survival (PFS), whilst high BRCA1 levels were associated with PFS on univariate analysis. With multivariate analyses no genes retained an association when adjusted by stage, grade and residual tumour. A tendency towards a better PFS was observed in patients with the highest level of ERCC1 and BRCA1 after platinum-based therapy than those given both platinum and taxol. The expression of DNA repair genes differed in borderline stage I, stage I and stage III ovarian carcinomas. The role of DNA repair genes in predicting the response in ovarian cancer patients seems far from being established. Copyright © 2010 Elsevier Ltd. All rights reserved.

  4. Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

    Science.gov (United States)

    Ewing, Margaret M; Thompson, Jonelle M; McLaren, Robert S; Purpero, Vincent M; Thomas, Kelli J; Dobrowski, Patricia A; DeGroot, Gretchen A; Romsos, Erica L; Storts, Douglas R

    2016-07-01

    Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation. Copyright © 2016 The Author(s). Published by Elsevier Ireland Ltd.. All rights reserved.

  5. Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without psoriasis

    DEFF Research Database (Denmark)

    Møller, P; Knudsen, Lisbeth E.; Frentz, G

    1998-01-01

    Quadruples of skin cancer patients with and without psoriasis and referents with and without psoriasis (4 x 20 study persons) were identified and examined for DNA damage by single cell gel electrophoresis (comet-assay) and DNA-repair by UV-induced unscheduled DNA synthesis (UDS) in mononuclear...... to solar radiation. When the comet tail moment data were stratified by sampling period, an interaction between psoriasis and skin cancer was detected, with patients with psoriasis and skin cancer exhibiting more DNA damage. Patients with psoriasis and skin cancer also had lower UDS compared to healthy...

  6. Multiple Determinations of Sperm DNA Fragmentation Show That Varicocelectomy Is Not Indicated for Infertile Patients with Subclinical Varicocele

    Directory of Open Access Journals (Sweden)

    Agustín García-Peiró

    2014-01-01

    Full Text Available Varicocele is one of the most common causes of low semen quality, which is reflected in high percentages of sperm cells with fragmented DNA. While varicocelectomy is usually performed to ameliorate a patient’s fertility, its impact on sperm DNA integrity in the case of subclinical varicocele is poorly documented. In this study, multiple DNA fragmentation analyses (TUNEL, SCD, and SCSA were performed on semen samples from sixty infertile patients with varicocele (15 clinical varicoceles, 19 clinical varicoceles after surgical treatment, 16 subclinical varicoceles, and 10 subclinical varicoceles after surgical treatment. TUNEL, SCD, and SCSA assays all showed substantial sperm DNA fragmentation levels that were comparable between subclinical and clinical varicocele patients. Importantly, varicocelectomy did improve sperm quality in patients with clinical varicocele; however, this was not the case in patients with subclinical varicocele. In summary, although infertile patients with clinical and subclinical varicocele have similar sperm DNA quality, varicocelectomy should only be advised for patients with clinical varicocele.

  7. Analysis of performance of a PCR-based assay to detect DNA of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical samples.

    Science.gov (United States)

    Bernal-Martínez, Leticia; Gago, Sara; Buitrago, María J; Gomez-Lopez, Alicia; Rodríguez-Tudela, Juan L; Cuenca-Estrella, Manuel

    2011-10-01

    The performance of a real-time PCR-based assay was retrospectively analyzed (according to European Organization for Research and Treatment of Cancer/Mycosis Study Group criteria) in the samples of patients with invasive aspergillosis. A total of 711 serial samples (356 whole-blood and 355 serum samples) from 38 adult patients were analyzed. The Aspergillus fumigatus PCR assay results were positive for 89 of 356 (25%) whole-blood samples and 90 of 355 (25.35%) serum samples. Positive PCR results were seen in 29 of 31 (93.5%) patients for which serum was analyzed and in 31 of 33 (93.9%) cases with whole-blood specimens. Both blood and serum samples were available in 26 cases, and significant differences were not observed in this subgroup of cases. The average number of threshold cycles (C(T)) for positive blood samples was 37.6, and the average C(T) for serum was 37.4. The DNA concentration ranged between 2 and 50 fg per μl of sample, with average DNA concentrations of 10.2 and 11.7 fg in positive blood and serum samples, respectively (P > 0.01). The performance of this PCR-based quantitative assay was similar for both serum and blood samples. We recommend serum samples as the most convenient hematological sample to use for Aspergillus DNA quantification when serial determinations are done.

  8. Estimation of serum concentration of parvovirus B19 DNA by PCR in patients with chronic anemia

    DEFF Research Database (Denmark)

    Hornsleth, A.; Carlsen, K. M.; Christensen, Laurids Siig

    1994-01-01

    Parvovirus B19 DNA was detected in serum samples from 10 out of 42 patients with chronic anaemia, the majority of whom suffered from aplastic anaemia, haemolytic anaemia, pure red cell anaemia or myelodysplastic syndrome. Nested PCR methods with sensitivities of 0.005-0.05 fg DNA were developed. ...

  9. Reproducibility of subgingival bacterial samples from patients with peri-implant mucositis

    DEFF Research Database (Denmark)

    Hallström, Hadar; Persson, G Rutger; Strömberg, Ulf

    2015-01-01

    collected with paper points and analyzed using the checkerboard DNA-DNA hybridization technique. Whole genomic probes of 74 preselected bacterial species were used. Based on the bacterial scores, Cohen's kappa coefficient was used to calculate the inter-annotator agreement for categorical data......OBJECTIVE: The aim of the present study was to investigate the reproducibility of bacterial enumeration from subsequent subgingival samples collected from patients with peri-implant mucositis. MATERIAL AND METHODS: Duplicate microbial samples from 222 unique implant sites in 45 adult subjects were....... The percentage agreement was considered as "good" when the two samples showed the same score or differed by 1 to the power of 10. RESULTS: Moderate to fair kappa values were displayed for all bacterial species in the test panel (range 0.21-0.58). There were no significant differences between Gram...

  10. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed samples compared to restored FFPE DNA

    DEFF Research Database (Denmark)

    Andersen, Gitte Brinch; Hager, Henrik; Hansen, Lise Lotte

    2014-01-01

    Chip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better......Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies......, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap...

  11. Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples

    Directory of Open Access Journals (Sweden)

    Ningxin Zhang

    2018-06-01

    Full Text Available The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS modification of the highly discriminatory C. albicans MLST (multilocus sequence typing method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type. Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to

  12. Serial monitoring of Mucorales DNA load in serum samples of a patient with disseminated mucormycosis after allogeneic bone marrow transplantation.

    Science.gov (United States)

    Shigemura, Tomonari; Nakazawa, Yozo; Matsuda, Kazuyuki; Sano, Kenji; Yaguchi, Takashi; Motobayashi, Mitsuo; Saito, Shoji; Noda, Shunsuke; Kobayashi, Norimoto; Agematsu, Kazunaga; Honda, Takayuki; Koike, Kenichi

    2014-08-01

    Mucormycosis is a fatal complication in immunocompromised patients, and is additionally difficult to diagnose due to the lack of useful serum biomarkers. Using a quantitative PCR approach, we retrospectively analyzed Mucorales DNA load in sera collected serially from a 3-year-old patient with chronic granulomatous disease, who died of multi-organ failure probably due to dissemination of Rhizomucor pusillus, which was detected from necropsy specimens. Mucorales DNA load was below the detection limit on days 9, 2, and 4 after unrelated bone marrow transplantation. Rhizomucor DNA was first detected on day 14 (1.6 × 10(3) copies/mL), and subsequently fluctuated between 1.3 × 10(3) and 37.2 × 10(3) copies/mL until day 43. Rhizomucor achieved a peak value of 940.0 × 10(3) copies/mL on day 48 the day before death. The detection or fluctuation of Rhizomucor DNA appeared to be associated with corticosteroid dosages or C-reactive protein levels. This specific, noninvasive, and highly quantitative assay may be useful for the early diagnosis of mucormycosis and prediction of disease progression.

  13. Detection of Toxoplasma gondii DNA in peripheral blood and aqueous humor of patients with Toxoplasmic active focal necrotizing retinochoroiditis using real-time PCR

    Directory of Open Access Journals (Sweden)

    Fabio Felipe dos Santos

    2015-12-01

    Full Text Available ABSTRACT Purpose: To evaluate the ability of real-time quantitative PCR (qPCR for detectingToxoplasma gondii DNA in the peripheral blood and aqueous humor of patients with toxoplasmic active focal necrotizing retinochoroiditis. Methods: Fifty-five patients with infectious uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were enrolled in this study. Forty-three patients had toxoplasmic active focal necrotizing retinochoroiditis, and the remaining 12 had non-toxoplasmic infectious uveitis and served as controls. qPCR analysis forT. gondii DNA was performed on the patients' peripheral blood and aqueous humor samples. Results: The qPCR was positive for T. gondii DNA in 37.21% (16/43 of the aqueous humor samples and 2.33% (1/43 of the peripheral blood samples; further, 16.27% (7/43 of the patients had positive results in both their blood and aqueous humor samples. Conclusion: qPCR was able to detect T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis in the blood as well as the aqueous humor and can help with the diagnosis of the disease.

  14. Monitoring of DNA and cytogenetic damage in lymphocytes in patients with skin cancer disease

    International Nuclear Information System (INIS)

    Cebulska-Wasilewska, A.; Dyga, W.; Krasnowolski, S.; Wierzewska, A.; Budzanowska, E.

    1999-01-01

    One major oncogenesis process is activation of proto-oncogenes by point mutations or chromosomal translocations. There is substantial evidence that indicates that human carcinogenesis involves loss of heterozygosity of certain chromosomes. Our study aimed at searching the possible association between cancer and DNA and cytogenetic abnormalities induced in lymphocytes of people with various categories of skin cancer cells. Fresh blood was collected by venepuncture from 25 individuals (including nine prior to cancer treatment). All patients were nonsmoking males, however 42.3% of them were former smokers. Blood samples were divided into two parts; in the first part of samples cytogenetic studies were performed immediately, while lymphocytes from the other part were isolated and stored at -70 0C for further studies in vitro. A single cell gel electrophoresis assay (SCGE), known as a Comet assay, was performed on them to study individual susceptibility to the induction of DNA damage by UV or radiation and to estimate variability in cellular repair capabilities. On average 220 good metaphase spreads per sample in the first mitotic division, and 100 spreads per sample in the second division were accepted for analysis of the cytogenetic damage. Chromosome and chromatid type aberrations were scored in the cells in the first mitosis, and expressed as total aberration frequency including and excluding gaps. Sister chromatid exchanges , high frequency cells and proliferating rate index were screened and evaluated in the second mitosis. Each patient showed a level exceeding (in at least one of the cytogenetic biomarker) the biomarker level in a reference group. In order to estimate susceptibility of people to environmentally induced damage, the isolated lymphocytes were irradiated with 2 Gy dose of X-rays or 6 J/m 2 of UV radiation, and the single cell gel electrophoresis (SCGE assay) was performed. To compare various individual capabilities to repair the induced damage

  15. Clinical Relevance of CDH1 and CDH13 DNA-Methylation in Serum of Cervical Cancer Patients

    Directory of Open Access Journals (Sweden)

    Günther K. Bonn

    2012-07-01

    Full Text Available This study was designed to investigate the DNA-methylation status of E-cadherin (CDH1 and H-cadherin (CDH13 in serum samples of cervical cancer patients and control patients with no malignant diseases and to evaluate the clinical utility of these markers. DNA-methylation status of CDH1 and CDH13 was analyzed by means of MethyLight-technology in serum samples from 49 cervical cancer patients and 40 patients with diseases other than cancer. To compare this methylation analysis with another technique, we analyzed the samples with a denaturing high performance liquid chromatography (DHPLC PCR-method. The specificity and sensitivity of CDH1 DNA-methylation measured by MethyLight was 75% and 55%, and for CDH13 DNA-methylation 95% and 10%. We identified a specificity of 92.5% and a sensitivity of only 27% for the CDH1 DHPLC-PCR analysis. Multivariate analysis showed that serum CDH1 methylation-positive patients had a 7.8-fold risk for death (95% CI: 2.2–27.7; p = 0.001 and a 92.8-fold risk for relapse (95% CI: 3.9–2207.1; p = 0.005. We concluded that the serological detection of CDH1 and CDH13 DNA-hypermethylation is not an ideal diagnostic tool due to low diagnostic specificity and sensitivity. However, it was validated that CDH1 methylation analysis in serum samples may be of potential use as a prognostic marker for cervical cancer patients.

  16. Genetic DNA profile in urine and hair follicles from patients who have undergone allogeneic hematopoietic stem cell transplantation.

    Science.gov (United States)

    Santurtún, Ana; Riancho, José A; Santurtún, Maite; Richard, Carlos; Colorado, M Mercedes; García Unzueta, Mayte; Zarrabeitia, María T

    2017-09-01

    Biological samples from patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) constitute a challenge for individual identification. In this study we analyzed the genetic profiles (by the amplification of 15 autosomic STRs) of HSCT patients found in different types of samples (blood, hair and urine) that may be the source of DNA in civil or criminal forensic cases. Our results show that while in hair follicles the donor component was not detected in any patient, thus being a reliable source of biological material for forensic identification, mixed chimerism was detected in urine samples from all patient, and no correlation was found between the time elapsed from the transplant and the percentage of chimerism. These results certainly have practical implications if the urine is being considered as a source of DNA for identification purposes in HSTC patients. Moreover, taking into consideration that chimerism was found not only in patients with leukocyturia (given the hematopoietic origin of leukocytes, this was expected), but also in those without observable leukocytes in the sediment, we conclude that an alternative source or sources of donor DNA must be implicated. Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.

  17. Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification.

    Science.gov (United States)

    Calacal, Gayvelline C; Apaga, Dame Loveliness T; Salvador, Jazelyn M; Jimenez, Joseph Andrew D; Lagat, Ludivino J; Villacorta, Renato Pio F; Lim, Maria Cecilia F; Fortun, Raquel D R; Datar, Francisco A; De Ungria, Maria Corazon A

    2015-11-01

    The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store. We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼ 1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA(®) cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor(®)HY. All samples were amplified using

  18. Mycobacterium ulcerans DNA not detected in faecal samples from Buruli ulcer patients: results of a pilot study.

    Directory of Open Access Journals (Sweden)

    Fred S Sarfo

    Full Text Available It has recently been shown that in a Buruli ulcer (BU endemic region of southeastern Australia, significant numbers of possums (native tree-dwelling marsupials have clinical BU disease. Furthermore, based on quantitative PCR (qPCR analysis, animals with BU lesions (and some without shed M. ulcerans DNA in their faeces, indicative of bacterial loads of up to 10(8 organisms/gram. These findings led us to propose that humans might also harbour M. ulcerans in their gastrointestinal tract and shed the bacterium in their faeces. We conducted a pilot study and collected faecal swabs from 26 patients with confirmed BU and 31 healthy household controls. Faecal samples were also collected from 10 healthy controls from non-endemic regions in Ghana. All 67 specimens were negative when tested by IS2404 PCR. The detection sensitivity of this method was ≥10(4 bacteria per gram (wet-weight of human faecal material. We conclude that the human gastrointestinal tract is unlikely to be a significant reservoir of M. ulcerans.

  19. Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification.

    Science.gov (United States)

    Poulsen, Nicklas N; Pedersen, Morten E; Østergaard, Jesper; Petersen, Nickolaj J; Nielsen, Christoffer T; Heegaard, Niels H H; Jensen, Henrik

    2016-09-20

    Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

  20. Impact of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA-DNA hybridization method.

    Science.gov (United States)

    do Nascimento, Cássio; dos Santos, Janine Navarro; Pedrazzi, Vinícius; Pita, Murillo Sucena; Monesi, Nadia; Ribeiro, Ricardo Faria; de Albuquerque, Rubens Ferreira

    2014-01-01

    Molecular diagnosis methods have been largely used in epidemiological or clinical studies to detect and quantify microbial species that may colonize the oral cavity in healthy or disease. The preservation of genetic material from samples remains the major challenge to ensure the feasibility of these methodologies. Long-term storage may compromise the final result. The aim of this study was to evaluate the effect of temperature and time storage on the microbial detection of oral samples by Checkerboard DNA-DNA hybridization. Saliva and supragingival biofilm were taken from 10 healthy subjects, aliquoted (n=364) and processed according to proposed protocols: immediate processing and processed after 2 or 4 weeks, and 6 or 12 months of storage at 4°C, -20°C and -80°C. Either total or individual microbial counts were recorded in lower values for samples processed after 12 months of storage, irrespective of temperatures tested. Samples stored up to 6 months at cold temperatures showed similar counts to those immediately processed. The microbial incidence was also significantly reduced in samples stored during 12 months in all temperatures. Temperature and time of oral samples storage have relevant impact in the detection and quantification of bacterial and fungal species by Checkerboard DNA-DNA hybridization method. Samples should be processed immediately after collection or up to 6 months if conserved at cold temperatures to avoid false-negative results. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Quantification of integrated HIV DNA by repetitive-sampling Alu-HIV PCR on the basis of poisson statistics.

    Science.gov (United States)

    De Spiegelaere, Ward; Malatinkova, Eva; Lynch, Lindsay; Van Nieuwerburgh, Filip; Messiaen, Peter; O'Doherty, Una; Vandekerckhove, Linos

    2014-06-01

    Quantification of integrated proviral HIV DNA by repetitive-sampling Alu-HIV PCR is a candidate virological tool to monitor the HIV reservoir in patients. However, the experimental procedures and data analysis of the assay are complex and hinder its widespread use. Here, we provide an improved and simplified data analysis method by adopting binomial and Poisson statistics. A modified analysis method on the basis of Poisson statistics was used to analyze the binomial data of positive and negative reactions from a 42-replicate Alu-HIV PCR by use of dilutions of an integration standard and on samples of 57 HIV-infected patients. Results were compared with the quantitative output of the previously described Alu-HIV PCR method. Poisson-based quantification of the Alu-HIV PCR was linearly correlated with the standard dilution series, indicating that absolute quantification with the Poisson method is a valid alternative for data analysis of repetitive-sampling Alu-HIV PCR data. Quantitative outputs of patient samples assessed by the Poisson method correlated with the previously described Alu-HIV PCR analysis, indicating that this method is a valid alternative for quantifying integrated HIV DNA. Poisson-based analysis of the Alu-HIV PCR data enables absolute quantification without the need of a standard dilution curve. Implementation of the CI estimation permits improved qualitative analysis of the data and provides a statistical basis for the required minimal number of technical replicates. © 2014 The American Association for Clinical Chemistry.

  2. Sample pretretment in microsystems

    DEFF Research Database (Denmark)

    Perch-Nielsen, Ivan R.

    2003-01-01

    : Sample preparation → DNA amplification → DNA analysis. The overall goal of the project is integration of as many as possible of these steps. This thesis covers mainly pretreatment in a microchip. Some methods for sample pretreatment have been tested. Most conventional is fluorescence activated cell sort......When a sample, e.g. from a patient, is processed using conventional methods, the sample must be transported to the laboratory where it is analyzed, after which the results is sent back. By integrating the separate steps of the analysis in a micro total analysis system (μTAS), results can...... be obtained fast and better. Preferably with all the processes from sample to signal moved to the bedside of the patient. Of course there is still much to learn and study in the process of miniaturization. DNA analysis is one process subject to integration. There are roughly three steps in a DNA analysis...

  3. Methods to maximise recovery of environmental DNA from water samples.

    Directory of Open Access Journals (Sweden)

    Rheyda Hinlo

    Full Text Available The environmental DNA (eDNA method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3-5 days. This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.

  4. Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases

    Science.gov (United States)

    Li, Yingmei; Pan, Wenying; Connolly, Ian D.; Reddy, Sunil; Nagpal, Seema

    2017-01-01

    Cerebral spinal fluid (CSF) from brain tumor patients contains tumor cellular and cell-free DNA (cfDNA), which provides a less-invasive and routinely accessible method to obtain tumor genomic information. In this report, we used droplet digital PCR to test mutant tumor DNA in CSF of a patient to monitor the treatment response of metastatic melanoma leptomeningeal disease (LMD). The primary melanoma was known to have a BRAFV600E mutation, and the patient was treated with whole brain radiotherapy and BRAF inhibitors. We collected 9 CSF samples over 6 months. The mutant cfDNA fraction gradually decreased from 53 % (time of diagnosis) to 0 (time of symptom alleviation) over the first 6 time points. Three months after clinical improvement, the patient returned with severe symptoms and the mutant cfDNA was again detected in CSF at high levels. The mutant DNA fraction corresponded well with the patient’s clinical response. We used whole exome sequencing to examine the mutation profiles of the LMD tumor DNA in CSF before therapeutic response and after disease relapse, and discovered a canonical cancer mutation PTENR130* at both time points. The cellular and cfDNA revealed similar mutation profiles, suggesting cfDNA is representative of LMD cells. This study demonstrates the potential of using cellular or cfDNA in CSF to monitor treatment response for LMD. PMID:26961773

  5. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund

    2015-01-01

    , are diverging due to methodological differences with lack of standardisation and definition of sensitivity. The new biological information has not yet come into routine use. The present study presents external standardisation by spiking with non-human DNA fragments to control for loss of DNA during sample...... preparation and measurement. It also suggests a method to control for admixture of DNA from normal lymphocytes by utilizing the unique immunoglobulin gene rearrangement in the B-cells. The results show that this approach improves the quality of the analysis and lowers the risk of falsely increased values...

  6. Y-STR analysis on DNA mixture samples--results of a collaborative project of the ENFSI DNA Working Group

    DEFF Research Database (Denmark)

    Parson, Walther; Niederstätter, Harald; Lindinger, Alexandra

    2008-01-01

    The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster C...... a laboratory-specific optimization process is indicated to reach a comparable sensitivity for the analysis of minute amounts of DNA....

  7. Reverse sample genome probing, a new technique for identification of bacteria in environmental samples by DNA hybridization, and its application to the identification of sulfate-reducing bacteria in oil field samples

    International Nuclear Information System (INIS)

    Voordouw, G.; Voordouw, J.K.; Karkhoff-Schweizer, R.R.; Fedorak, P.M.; Westlake, D.W.S.

    1991-01-01

    A novel method for identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a standard) to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples

  8. Pengujian Kartu Fta Sebagai Alat Sampling Dna Jamur Patogen Dariberbagi Bagian Tanaman Yang Terinfeksi

    OpenAIRE

    Sulistyawati, Purnamila

    2011-01-01

    FTA card offers a simple and fast method for retrieval of DNA samples at room temperature and storage of DNA in the short and long term. This will fasiliate the detection and identification of plant pathogens rapidly; increasing the number of samples can be collected, stored and transported in the field, especially from remote locations. The purpose of this study is to investigate the suitability of FTA cards as a new method for sampling DNA from multiple infected of plant tissues such as ...

  9. Evaluation of Patients with an Apparent False Positive Stool DNA Test: The Role of Repeat Stool DNA Testing.

    Science.gov (United States)

    Cooper, Gregory S; Markowitz, Sanford D; Chen, Zhengyi; Tuck, Missy; Willis, Joseph E; Berger, Barry M; Brenner, Dean E; Li, Li

    2018-03-07

    There is uncertainty as to the appropriate follow-up of patients who test positive on multimarker stool DNA (sDNA) testing and have a colonoscopy without neoplasia. To determine the prevalence of missed colonic or occult upper gastrointestinal neoplasia in patients with an apparent false positive sDNA. We prospectively identified 30 patients who tested positive with a commercially available sDNA followed by colonoscopy without neoplastic lesions. Patients were invited to undergo repeat sDNA at 11-29 months after the initial test followed by repeat colonoscopy and upper endoscopy. We determined the presence of neoplastic lesions on repeat evaluation stratified by results of repeat sDNA. Twelve patients were restudied. Seven patients had a negative second sDNA test and a normal second colonoscopy and upper endoscopy. In contrast, 5 of 12 subjects had a persistently positive second sDNA test, and 3 had positive findings, including a 3-cm sessile transverse colon adenoma with high-grade dysplasia, a 2-cm right colon sessile serrated adenoma with dysplasia, and a nonadvanced colon adenoma (p = 0.045). These corresponded to a positive predictive value of 0.60 (95% CI 0.17-1.00) and a negative predictive value of 1.00 (95% CI 1.00-1.00) for the second sDNA test. In addition, the medical records of all 30 subjects with apparent false positive testing were reviewed and no documented cases of malignant tumors were recorded. Repeat positive sDNA testing may identify a subset of patients with missed or occult colorectal neoplasia after negative colonoscopy for an initially positive sDNA. High-quality colonoscopy with careful attention to the right colon in patients with positive sDNA is critically important and may avoid false negative colonoscopy.

  10. Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2012-01-01

    Abstract DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above...... data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated....

  11. Dynamic In Vivo Profiling of DNA Damage and Repair after Radiotherapy Using Canine Patients as a Model

    Directory of Open Access Journals (Sweden)

    Nadine Schulz

    2017-06-01

    Full Text Available Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX immunohistochemistry. In vivo results were then compared (in silico with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15–60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.

  12. Correlation of MLH1 and MGMT methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients.

    Science.gov (United States)

    Li, Xia; Wang, Yibaina; Zhang, Zuoming; Yao, Xiaoping; Ge, Jie; Zhao, Yashuang

    2013-11-01

    CpG island methylation in the promoter regions of the DNA mismatch repair gene mutator L homologue 1 ( MLH1 ) and DNA repair gene O 6 -methylguanine-DNA methyltransferase ( MGMT ) genes has been shown to occur in the leukocytes of peripheral blood and colorectal tissue. However, it is unclear whether the methylation levels in the blood leukocytes and colorectal tissue are correlated. The present study analyzed and compared the levels of MGMT and MLH1 gene methylation in the leukocytes of peripheral blood and colorectal tissues obtained from patients with colorectal cancer (CRC). The methylation levels of MGMT and MLH1 were examined using methylation-sensitive high-resolution melting (MS-HRM) analysis. A total of 44 patients with CRC were selected based on the MLH1 and MGMT gene methylation levels in the leukocytes of the peripheral blood. Corresponding colorectal tumor and normal tissues were obtained from each patient and the DNA methylation levels were determined. The correlation coefficients were evaluated using Spearman's rank test. Agreement was determined by generalized κ-statistics. Spearman's rank correlation coefficients (r) for the methylation levels of the MGMT and MLH1 genes in the leukocytes of the peripheral blood and normal colorectal tissue were 0.475 and 0.362, respectively (P=0.001 and 0.016, respectively). The agreement of the MGMT and MLH1 gene methylation levels in the leukocytes of the peripheral blood and normal colorectal tissue were graded as fair and poor (κ=0.299 and 0.126, respectively). The methylation levels of MGMT and MLH1 were moderately and weakly correlated between the patient-matched leukocytes and the normal colorectal tissue, respectively. Blood-derived DNA methylation measurements may not always represent the levels of normal colorectal tissue methylation.

  13. Inspecting close maternal relatedness: Towards better mtDNA population samples in forensic databases.

    Science.gov (United States)

    Bodner, Martin; Irwin, Jodi A; Coble, Michael D; Parson, Walther

    2011-03-01

    Reliable data are crucial for all research fields applying mitochondrial DNA (mtDNA) as a genetic marker. Quality control measures have been introduced to ensure the highest standards in sequence data generation, validation and a posteriori inspection. A phylogenetic alignment strategy has been widely accepted as a prerequisite for data comparability and database searches, for forensic applications, for reconstructions of human migrations and for correct interpretation of mtDNA mutations in medical genetics. There is continuing effort to enhance the number of worldwide population samples in order to contribute to a better understanding of human mtDNA variation. This has often lead to the analysis of convenience samples collected for other purposes, which might not meet the quality requirement of random sampling for mtDNA data sets. Here, we introduce an additional quality control means that deals with one aspect of this limitation: by combining autosomal short tandem repeat (STR) marker with mtDNA information, it helps to avoid the bias introduced by related individuals included in the same (small) sample. By STR analysis of individuals sharing their mitochondrial haplotype, pedigree construction and subsequent software-assisted calculation of likelihood ratios based on the allele frequencies found in the population, closely maternally related individuals can be identified and excluded. We also discuss scenarios that allow related individuals in the same set. An ideal population sample would be representative for its population: this new approach represents another contribution towards this goal. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  14. Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

    Directory of Open Access Journals (Sweden)

    Mayra Eduardoff

    2017-09-01

    Full Text Available The analysis of mitochondrial DNA (mtDNA has proven useful in forensic genetics and ancient DNA (aDNA studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR is commonly sequenced using established Sanger-type Sequencing (STS protocols involving fragment sizes down to approximately 150 base pairs (bp. Recent developments include Massively Parallel Sequencing (MPS of (multiplex PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less, and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples, and tested challenging forensic samples (n = 2 as well as compromised solid tissue samples (n = 15 up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS

  15. DNA Superresolution Structure of Reed-Sternberg Cells Differs Between Long-Lasting Remission Versus Relapsing Hodgkin's Lymphoma Patients.

    Science.gov (United States)

    Righolt, Christiaan H; Knecht, Hans; Mai, Sabine

    2016-07-01

    Recent developments in microscopy have led to superresolution microscopy images of cells. Structured illumination microscopy was used before to reveal new details in the DNA structure and the structure of the DNA-free space in the DAPI-stained cell nuclei of the Hodgkin's lymphoma HDLM-2 cell line. This study extends this technology to primary pre-treatment classical Hodgkin's lymphoma samples of ten patients. Significant differences in both the DNA structure and the structure of the DNA-free space were detected between lymphocytes and malignant cells. Both types of structures were similar for lymphocytes of different patients. When the patients were un-blinded and grouped based on their clinical outcome, either non-relapsed or relapsed, a significant difference in the DNA structure of their Reed-Sternberg (RS) cells was found. Since, RS cells develop from mono-nucleated Hodgkin (H) cells, these data suggest distinct architectural restructuring of nuclei during RS cell formation in patients going to long-lasting remission versus relapse. J. Cell. Biochem. 117: 1633-1637, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  16. Statistical model for degraded DNA samples and adjusted probabilities for allelic drop-out

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2012-01-01

    DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above a certain...... data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated....

  17. Paleoparasitological report on Ascaris aDNA from an ancient East Asian sample

    Directory of Open Access Journals (Sweden)

    Chang Seok Oh

    2010-03-01

    Full Text Available In this study, Ascaris DNA was extracted and sequenced from a medieval archaeological sample in Korea. While Ascaris eggs were confirmed to be of human origin by archaeological evidence, it was not possible to pinpoint the exact species due to close genetic relationships among them. Despite this shortcoming, this is the first Ascaris ancient DNA (aDNA report from a medieval Asian country and thus will expand the scope of Ascaris aDNA research.

  18. Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

    Science.gov (United States)

    Kalmár, Alexandra; Péterfia, Bálint; Hollósi, Péter; Wichmann, Barnabás; Bodor, András; Patai, Árpád V; Schöller, Andrea; Krenács, Tibor; Tulassay, Zsolt; Molnár, Béla

    2015-09-01

    We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

  19. Environmental DNA from Seawater Samples Correlate with Trawl Catches of Subarctic, Deepwater Fishes.

    Directory of Open Access Journals (Sweden)

    Philip Francis Thomsen

    Full Text Available Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA metabarcoding of seawater samples from continental slope depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites showed good correspondence with catch sizes. Environmental DNA sequence reads from the fish assemblages correlated with biomass and abundance data obtained from trawling. Interestingly, the Greenland shark (Somniosus microcephalus showed high abundance of eDNA reads despite only a single specimen being caught, demonstrating the relevance of the eDNA approach for large species that can probably avoid bottom trawls in most cases. Quantitative detection of marine fish using eDNA remains to be tested further to ascertain whether this technique is able to yield credible results for routine application in fisheries. Nevertheless, our study demonstrates that eDNA reads can be used as a qualitative and quantitative proxy for marine fish assemblages in deepwater oceanic

  20. The efficacy of 16S ribosomal DNA sequencing in the diagnosis of bacteria from blood, bone and synovial fluid samples of children with musculoskeletal infections.

    Science.gov (United States)

    Hashavya, S; Gross, I; Michael-Gayego, A; Simanovsky, N; Lamdan, R

    2018-04-01

    Musculoskeletal infections are among the most common bacterial infections in children leading to hospitalization, invasive procedures and prolonged antibiotic administration. Blood, synovial and sometimes tissue cultures are essential for the diagnosis and treatment of musculoskeletal infections; 16S ribosomal DNA (rDNA) sequencing is a novel diagnostic tool for the detection of bacteria.While the yield of 16S rDNA sequencing in synovial fluid was previously assessed, data regarding the efficacy of this method from blood samples or partially treated children with suspected musculoskeletal infections is lacking.In this study we assessed the yield of 16S rDNA sequencing in blood, bone and synovial samples of children with musculoskeletal infections. Blood, synovial and bone samples were collected from children with suspected musculoskeletal infections and analyzed for the presence of 16S rDNA, the results were then compared with the benchmark microbial cultures. During the study period, 41 children (18 boys and 23 girls) with suspected acute musculoskeletal infection were enrolled. A positive blood culture was found in 6/31 cases (19.4%) with methicillin-susceptible Staphylococcus aureus being the most commonly isolated bacterium. No significant 16S rDNA detection in blood samples was recorded.Synovial fluid culture was positive in 6/28 samples (21%), Kingella kingae being the most common pathogen. When using the 16S rDNA sequencing method, the rate of positive results in synovial fluid was higher with bacterial detection in 12/23 (52%) samples. The 16S rDNA sequencing method was also able to identify pathogens in samples taken from partially treated children where cultures were negative with 16S rDNA detection in 5/5 samples. Although 16S rDNA sequencing may increase the yield of bacterial detection in synovial samples of patients with musculoskeletal infections, there is no benefit from applying this method on blood samples. The 16S rDNA sequencing method may be

  1. Detection of Circulating Mucorales DNA in Critically Ill Burn Patients: Preliminary Report of a Screening Strategy for Early Diagnosis and Treatment.

    Science.gov (United States)

    Legrand, Matthieu; Gits-Muselli, Maud; Boutin, Louis; Garcia-Hermoso, Dea; Maurel, Véronique; Soussi, Sabri; Benyamina, Mourad; Ferry, Axelle; Chaussard, Maïté; Hamane, Samia; Denis, Blandine; Touratier, Sophie; Guigue, Nicolas; Fréalle, Emilie; Jeanne, Mathieu; Shaal, Jean-Vivien; Soler, Charles; Mimoun, Maurice; Chaouat, Marc; Lafaurie, Matthieu; Mebazaa, Alexandre; Bretagne, Stéphane; Alanio, Alexandre

    2016-11-15

     Invasive wound mucormycosis (IWM) is associated with an extremely poor outcome among critically ill burn patients. We describe the detection of circulating Mucorales DNA (cmDNA) for the early diagnosis of IWM in those patients and report the potential value of detecting cmDNA for treatment guidance.  Severely ill burn patients admitted to our tertiary referral center between October 2013 and February 2016 were included. Retrospective plasma samples were tested for the presence of cmDNA by quantitative real-time polymerase chain reaction (qPCR). Patients were then prospectively screened twice a week, and liposomal amphotericin-B therapy initiated based on a positive qPCR. The primary endpoint was the time between cmDNA detection and standard diagnosis. Secondary endpoints were the time from cmDNA detection and treatment initiation and mortality.  Seventy-seven patients (418 samples) were included. The average age was 46 (28-60) years, abbreviated burn severity index was 8 (7-10), and simplified acute physiology score was 33 (23-46). The total body surface area was 33% (22%-52%). cmDNA was detected 11 (4.5-15) days before standard diagnosis. The in-hospital mortality was 62% for patients with IWM and 24% for those without (P = .03). The mortality due to IWM was 80% during period A and 33% during period B (P = .46).  This study suggests that the detection of cmDNA allows earlier diagnosis of IWM in severely ill burn patients and earlier initiation of treatment. Further studies are needed to confirm the impact of earlier treatment initiation on patient outcome. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

  2. LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples.

    Science.gov (United States)

    Deb, R; Sengar, G S; Singh, U; Kumar, S; Raja, T V; Alex, R; Alyethodi, R R; Prakash, B

    2017-01-01

    Animal species detection is one of the crucial steps for consumer's food analysis. In the present study we developed an in-house built loop-mediated isothermal amplification (LAMP) assay for rapid detection of adulterated cow DNA in goat milk/meat samples. The cow milk/tissue DNA in goat milk/meat samples were identified in the developed LAMP assay by either naked eye visualizing with SYBR Green I dyes or by detecting the typical ladder pattern on gel electrophoresis. This test can detect up to minimum 5% level of cow components admixed in goat milk/meat samples and can be completed within 1 h 40 min starting from DNA extraction from milk/meat samples and can be performed in a water bath. Developed LAMP methodology is simple; rapid and sensitive techniques that can detect adulterant like cow components in goat milk/meat are more accurate than other existing DNA based technologies.

  3. Circulatory mitochondrial DNA is a pro-inflammatory agent in maintenance hemodialysis patients.

    Directory of Open Access Journals (Sweden)

    Hongdi Cao

    Full Text Available Chronic inflammation is highly prevalent in maintenance hemodialysis (MHD patients, and it has been shown to be a strong predictor of morbidity and mortality. Mitochondrial DNA (mtDNA released into circulation after cell damage can promote inflammation in patients and animal models. However, the role and mechanisms of circulatory mtDNA in chronic inflammation in MHD patients remain unknown. Sixty MHD patients and 20 health controls were enrolled in this study. The circulatory mtDNA was detected by quantitative real-time PCR assay. Plasma interleukin 6 (IL-6 and tumor necrosis factor α (TNF-α were quantitated by ELISA assay. Dialysis systems in MHD patients and in vitro were used to evaluate the effect of different dialysis patterns on circulatory mtDNA. Circulatory mtDNA was elevated in MHD patients comparing to that of health control. Regression analysis demonstrated that plasma mtDNA was positively associated with TNF-α and the product of serum calcium and phosphorus, while negatively associated with hemoglobin and serum albumin in MHD patients. MtDNA induced the secretion of IL-6 and TNF-α in the THP-1 cells. Single high-flux hemodialysis (HF-HD and on line hemodiafiltration (OL-HDF but not low-flux hemodialysis (LF-HD could partially reduce plasma mtDNA in MHD patients. In vitro, both HD and hemofiltration (HF could fractional remove mtDNA. Collectively, circulatory mtDNA is elevated and its level is closely correlated with chronic inflammation in MHD patients. HF-HD and HDF can partially reduce circulatory mtDNA in MHD patients.

  4. Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy

    Science.gov (United States)

    Ling, Paul D.; Vilchez, Regis A.; Keitel, Wendy A.; Poston, David G.; Peng, Rong Sheng; White, Zoe S.; Visnegarwala, Fehmida; Lewis, Dorothy E.; Butel, Janet S.

    2003-01-01

    Patients with human immunodeficiency virus type 1 (HIV-1) infection are at high risk of developing Epstein-Barr virus (EBV)-associated lymphoma. However, little is known of the EBV DNA loads in patients receiving highly active antiretroviral therapy (HAART). Using a real-time quantitative polymerase chain reaction assay, we demonstrated that significantly more HIV-1-infected patients receiving HAART than HIV-1-uninfected volunteers had detectable EBV DNA in blood (57 [81%] of 70 vs. 11 [16%] of 68 patients; P=.001) and saliva (55 [79%] of 68 vs. 37 [54%] of 68 patients; P=.002). The mean EBV loads in blood and saliva samples were also higher in HIV-1-infected patients than in HIV-1-uninfected volunteers (P=.001). The frequency of EBV detection in blood was associated with lower CD4+ cell counts (P=.03) among HIV-1-infected individuals, although no differences were observed in the EBV DNA loads in blood or saliva samples in the HIV-1-infected group. Additional studies are needed to determine whether EBV-specific CD4+ and CD8+ cells play a role in the pathogenesis of EBV in HIV-1-infected patients receiving HAART.

  5. Filtration recovery of extracellular DNA from environmental water samples

    Science.gov (United States)

    qPCR methods are able to analyze DNA from microbes within hours of collecting water samples, providing the promptest notification and public awareness possible when unsafe pathogenic levels are reached. Health risk, however, may be overestimated by the presence of extracellular ...

  6. Compound mitochondrial DNA mutations in a neurological patient ...

    Indian Academy of Sciences (India)

    Compound mitochondrial DNA mutations in a neurological patient with ataxia, myoclonus and deafness. Ji Hoon Park, Bo Ram Yoon, Hye Jin Kim, Phil Hyu Lee, Byung-Ok Choi and Ki Wha Chung. J. Genet. 93, 173–177. Table 1. Variations from the whole mtDNA sequence in the AMDF patient. Mutation. Report. Locus/ ...

  7. Association of anti C1q and ds-DNA levels with the pathogenesis of Lupus Nephritis among SLE patients

    Directory of Open Access Journals (Sweden)

    Zara Sohail

    2018-02-01

    Full Text Available Background: Lupus nephritis (LN is the most common and serious complication associated with SLE and it results in significant morbidity and mortality. It is known by several studies that patients of LN have higher levels of anti-dsDNA and anti-C1q compared with SLE patients without renal involvement. The current study was designed to determine and compare the level of anti-dsDNA and anti-C1q in patients of SLE with and without lupus nephritis in the Pakistani population. This current study was also aimed at providing proof that anti-C1q levels are more prominent in LN/non-LN SLE as compared to anti-dsDNA. This project may help in the determination of results in Pakistan and contribute to the further confirmation of the sensitivity of anti-C1q. Method: The patient samples were collected from Sheikh Zayed hospital, Lahore. These patients were clinically diagnosed by the Rheumatologists as SLE and LN positive on the basis of ACR and SLEDAI scoring criteria. This study was performed and samples were analyzed in the Department of Medical and Laboratory Sciences, Imperial College of Business Study, Lahore on the patient’s serum by ELISA technique. Result: About 38% (12 patients with LN were positive for anti-dsDNA and 31% (9 SLE patients without LN were positive whereas about 38.7% (12 were anti-dsDNA negative in LN cases and 58.6% (17 in SLE without LN. In case of anti-C1q 100% (31 of these LN patients were positive and 93.1% (27 patients SLE without LN showed positive anti C1q results. Only 6.9% (2 patients showed negative results for anti-C1q in LN negative patients Conclusion: The higher levels of anti-C1q suggest that it may be a better diagnostic marker for LN than that of anti-dsDNA and that it can be helpful in the prognosis of SLE patients.

  8. Protocol for collecting eDNA samples from streams [Version 2.3

    Science.gov (United States)

    K. J. Carim; T. Wilcox; M. K. Young; K. S. McKelvey; M. K. Schwartz

    2015-01-01

    Throughout the 2014 field season, we had over two dozen biologist throughout the western US collect over 300 samples for eDNA analysis with paired controls. Control samples were collected by filtering 0.5 L of distilled water. No samples had any evidence of field contamination. This method of sampling verifies the cleanliness of the field equipment, as well as the...

  9. [The use of 16S rDNA sequencing in species diversity analysis for sputum of patients with ventilator-associated pneumonia].

    Science.gov (United States)

    Yang, Xiaojun; Wang, Xiaohong; Liang, Zhijuan; Zhang, Xiaoya; Wang, Yanbo; Wang, Zhenhai

    2014-05-01

    To study the species and amount of bacteria in sputum of patients with ventilator-associated pneumonia (VAP) by using 16S rDNA sequencing analysis, and to explore the new method for etiologic diagnosis of VAP. Bronchoalveolar lavage sputum samples were collected from 31 patients with VAP. Bacterial DNA of the samples were extracted and identified by polymerase chain reaction (PCR). At the same time, sputum specimens were processed for routine bacterial culture. The high flux sequencing experiment was conducted on PCR positive samples with 16S rDNA macro genome sequencing technology, and sequencing results were analyzed using bioinformatics, then the results between the sequencing and bacteria culture were compared. (1) 550 bp of specific DNA sequences were amplified in sputum specimens from 27 cases of the 31 patients with VAP, and they were used for sequencing analysis. 103 856 sequences were obtained from those sputum specimens using 16S rDNA sequencing, yielding approximately 39 Mb of raw data. Tag sequencing was able to inform genus level in all 27 samples. (2) Alpha-diversity analysis showed that sputum samples of patients with VAP had significantly higher variability and richness in bacterial species (Shannon index values 1.20, Simpson index values 0.48). Rarefaction curve analysis showed that there were more species that were not detected by sequencing from some VAP sputum samples. (3) Analysis of 27 sputum samples with VAP by using 16S rDNA sequences yielded four phyla: namely Acitinobacteria, Bacteroidetes, Firmicutes, Proteobacteria. With genus as a classification, it was found that the dominant species included Streptococcus 88.9% (24/27), Limnohabitans 77.8% (21/27), Acinetobacter 70.4% (19/27), Sphingomonas 63.0% (17/27), Prevotella 63.0% (17/27), Klebsiella 55.6% (15/27), Pseudomonas 55.6% (15/27), Aquabacterium 55.6% (15/27), and Corynebacterium 55.6% (15/27). (4) Pyrophosphate sequencing discovered that Prevotella, Limnohabitans, Aquabacterium

  10. Mutation of mtDNA ND1 Gene in 20 Type 2 Diabetes Mellitus Patients of Gorontalonese and Javanese Ethnicity

    Directory of Open Access Journals (Sweden)

    AMIEN RAMADHAN ISHAK

    2014-12-01

    Full Text Available Mitochondrial gene mutation plays a role in the development of type two diabetes mellitus (T2DM. A point mutation in the mitochondrial gene Nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND1 gene mainly reported as the most common mutation related to T2DM. However, several studies have identified another SNP (single-nucleotide polymorphisms in the RNA region of mtDNA from patients from specific ethnic populations in Indonesia. Building on those findings, this study aimed to use PCR and DNA sequencing technology to identify nucleotides in RNA and ND1 fragment from 20 Gorontalonese and 20 Javanese T2DM patients, that may trigger T2DM expression. The results showed successful amplification of RNA along 294 bp for all samples. From these samples, we found two types of point mutation in Javanese patients in the G3316A and T3200C points of the rRNA and ND1 gene. In samples taken from Gorontalonese patients, no mutation were found in the RNA or ND1 region. We conclude that T2DM was triggered differently in our two populations. While genetic mutation is implicated for the 20 Javanese patients, T2DM pathogenesis in the Gorontalonese patients must be traced to other genetic, environmental, or behavioral factors.

  11. Resolution of a serum sample mix-up through the use of short tandem repeat DNA typing.

    Science.gov (United States)

    Allen, Robert W; Pritchard, Jane K

    2004-12-01

    A sample mix-up occurred in a tissue procurement laboratory in which aliquots of serum from two tissue donors were accidentally mislabeled. The clues to the apparent mixup involved discrepant Hepatitis C test results. In an attempt to resolve the apparent mix up, DNA typing was performed using serum samples as a possible source of genomic DNA. Two hundred microliter aliquots of two reference sera and aliquots prepared from them were subjected to DNA extraction. PCR amplification of 9 STR loci was performed on the extracts and amplicons were analyzed by capillary electrophoresis. About 1 microg/ml of DNA was recovered from all serum samples and was of sufficient quality to direct the amplification of most, if not all STR loci allowing the mislabeled specimens to be traced to the proper tissue donor. Serum is a useful source of genomic DNA for STR analysis in situations in which such samples are the only source of DNA for testing. Interestingly, one of the tissue donors on life support and repeatedly receiving blood products, exhibited a mixed DNA profile indicative of the presence of DNA from multiple individuals in the bloodstream.

  12. A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples.

    Science.gov (United States)

    Guha, Pokhraj; Das, Avishek; Dutta, Somit; Chaudhuri, Tapas Kumar

    2018-01-01

    Different methods available for extraction of human genomic DNA suffer from one or more drawbacks including low yield, compromised quality, cost, time consumption, use of toxic organic solvents, and many more. Herein, we aimed to develop a method to extract DNA from 500 μL of fresh or frozen human blood. Five hundred microliters of fresh and frozen human blood samples were used for standardization of the extraction procedure. Absorbance at 260 and 280 nm, respectively, (A 260 /A 280 ) were estimated to check the quality and quantity of the extracted DNA sample. Qualitative assessment of the extracted DNA was checked by Polymerase Chain reaction and double digestion of the DNA sample. Our protocol resulted in average yield of 22±2.97 μg and 20.5±3.97 μg from 500 μL of fresh and frozen blood, respectively, which were comparable to many reference protocols and kits. Besides yielding bulk amount of DNA, our protocol is rapid, economical, and avoids toxic organic solvents such as Phenol. Due to unaffected quality, the DNA is suitable for downstream applications. The protocol may also be useful for pursuing basic molecular researches in laboratories having limited funds. © 2017 Wiley Periodicals, Inc.

  13. Species-specific identification from incomplete sampling: applying DNA barcodes to monitoring invasive solanum plants.

    Science.gov (United States)

    Zhang, Wei; Fan, Xiaohong; Zhu, Shuifang; Zhao, Hong; Fu, Lianzhong

    2013-01-01

    Comprehensive sampling is crucial to DNA barcoding, but it is rarely performed because materials are usually unavailable. In practice, only a few rather than all species of a genus are required to be identified. Thus identification of a given species using a limited sample is of great importance in current application of DNA barcodes. Here, we selected 70 individuals representing 48 species from each major lineage of Solanum, one of the most species-rich genera of seed plants, to explore whether DNA barcodes can provide reliable specific-species discrimination in the context of incomplete sampling. Chloroplast genes ndhF and trnS-trnG and the nuclear gene waxy, the commonly used markers in Solanum phylogeny, were selected as the supplementary barcodes. The tree-building and modified barcode gap methods were employed to assess species resolution. The results showed that four Solanum species of quarantine concern could be successfully identified through the two-step barcoding sampling strategy. In addition, discrepancies between nuclear and cpDNA barcodes in some samples demonstrated the ability to discriminate hybrid species, and highlights the necessity of using barcode regions with different modes of inheritance. We conclude that efficient phylogenetic markers are good candidates as the supplementary barcodes in a given taxonomic group. Critically, we hypothesized that a specific-species could be identified from a phylogenetic framework using incomplete sampling-through this, DNA barcoding will greatly benefit the current fields of its application.

  14. Developmental validation of the Quantifiler(®) HP and Trio Kits for human DNA quantification in forensic samples.

    Science.gov (United States)

    Holt, Allison; Wootton, Sharon Chao; Mulero, Julio J; Brzoska, Pius M; Langit, Emanuel; Green, Robert L

    2016-03-01

    The quantification of human genomic DNA is a necessary first step in the DNA casework sample analysis workflow. DNA quantification determines optimal sample input amounts for subsequent STR (short tandem repeat) genotyping procedures, as well as being a useful screening tool to identify samples most likely to provide probative genotypic evidence. To better mesh with the capabilities of newest-generation STR analysis assays, the Quantifiler(®) HP and Quantifiler(®) Trio DNA Quantification Kits were designed for greater detection sensitivity and more robust performance with samples that contain PCR inhibitors or degraded DNA. The new DNA quantification kits use multiplex TaqMan(®) assay-based fluorescent probe technology to simultaneously quantify up to three human genomic targets, allowing samples to be assessed for total human DNA, male contributor (i.e., Y-chromosome) DNA, as well as a determination of DNA degradation state. The Quantifiler HP and Trio Kits use multiple-copy loci to allow for significantly improved sensitivity compared to earlier-generation kits that employ single-copy target loci. The kits' improved performance provides better predictive ability for results with downstream, newest-generation STR assays, and their shortened time-to-result allows more efficient integration into the forensic casework analysis workflow. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Usefulness of in-house real time PCR for HBV DNA quantification in serum and oral fluid samples.

    Science.gov (United States)

    Portilho, Moyra Machado; Mendonça, Ana Carolina da Fonseca; Bezerra, Cristianne Sousa; do Espirito-Santo, Márcia Paschoal; de Paula, Vanessa Salete; Nabuco, Leticia Cancella; Villela-Nogueira, Cristiane Alves; Lewis-Ximenez, Lia Laura; Lampe, Elisabeth; Villar, Livia Melo

    2018-06-01

    For quantification of hepatitis B virus DNA (HBV DNA), commercial assays are used with serum or plasma samples, but oral fluid samples could be an alternative for HBV diagnosis due to ease of collection. This study aims to develop in-house real time PCR using synthetic curve for HBV DNA quantification for serum and oral fluid samples. Samples were collected from 103 individuals (55 HBsAg reactive and HBV DNA reactive by commercial assay and 48 without HBV markers) and submitted to two in-house real time PCR assays for HBV pre-S/S region with different standard curves: qPCR plasmidial and qPCR synthetic. A total of 27 serum samples were HBV DNA positive by qPCR plasmidial and 40 with qPCR synthetic (72% and 85% of concordance, respectively). Quantitative PCR synthetic presented efficiency of 99% and sensitivity of 2log10 copies/mL. Among oral fluid samples, five and ten were detected using qPCR plasmidial and synthetic, respectively. This study demonstrated that qPCR synthetic using serum samples could be used as alternative for HBV DNA quantification due to its sensitivity. In addition, it was possible to quantify HBV DNA in oral fluid samples suggesting the potential of this specimen for molecular diagnosis of HBV. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

    Science.gov (United States)

    Sidstedt, Maja; Romsos, Erica L; Hedell, Ronny; Ansell, Ricky; Steffen, Carolyn R; Vallone, Peter M; Rådström, Peter; Hedman, Johannes

    2017-02-07

    Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

  17. Sterile paper points as a bacterial DNA-contamination source in microbiome profiles of clinical samples

    NARCIS (Netherlands)

    van der Horst, J.; Buijs, M.J.; Laine, M.L.; Wismeijer, D.; Loos, B.G.; Crielaard, W.; Zaura, E.

    2013-01-01

    Objectives High throughput sequencing of bacterial DNA from clinical samples provides untargeted, open-ended information on the entire microbial community. The downside of this approach is the vulnerability to DNA contamination from other sources than the clinical sample. Here we describe

  18. Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

    Science.gov (United States)

    Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

    2018-01-01

    We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

  19. Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples

    International Nuclear Information System (INIS)

    Sayler, G.S.; Shields, M.S.; Tedford, E.T.; Breen, A.; Hooper, S.W.; Sirotkin, K.M.; Davis, J.W.

    1985-01-01

    The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations

  20. Using CF11 cellulose columns to inexpensively and effectively remove human DNA from Plasmodium falciparum-infected whole blood samples

    Directory of Open Access Journals (Sweden)

    Venkatesan Meera

    2012-02-01

    Full Text Available Abstract Background Genome and transcriptome studies of Plasmodium nucleic acids obtained from parasitized whole blood are greatly improved by depletion of human DNA or enrichment of parasite DNA prior to next-generation sequencing and microarray hybridization. The most effective method currently used is a two-step procedure to deplete leukocytes: centrifugation using density gradient media followed by filtration through expensive, commercially available columns. This method is not easily implemented in field studies that collect hundreds of samples and simultaneously process samples for multiple laboratory analyses. Inexpensive syringes, hand-packed with CF11 cellulose powder, were recently shown to improve ex vivo cultivation of Plasmodium vivax obtained from parasitized whole blood. This study was undertaken to determine whether CF11 columns could be adapted to isolate Plasmodium falciparum DNA from parasitized whole blood and achieve current quantity and purity requirements for Illumina sequencing. Methods The CF11 procedure was compared with the current two-step standard of leukocyte depletion using parasitized red blood cells cultured in vitro and parasitized blood obtained ex vivo from Cambodian patients with malaria. Procedural variations in centrifugation and column size were tested, along with a range of blood volumes and parasite densities. Results CF11 filtration reliably produces 500 nanograms of DNA with less than 50% human DNA contamination, which is comparable to that obtained by the two-step method and falls within the current quality control requirements for Illumina sequencing. In addition, a centrifuge-free version of the CF11 filtration method to isolate P. falciparum DNA at remote and minimally equipped field sites in malaria-endemic areas was validated. Conclusions CF11 filtration is a cost-effective, scalable, one-step approach to remove human DNA from P. falciparum-infected whole blood samples.

  1. Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

    Science.gov (United States)

    Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

    2006-08-14

    Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to

  2. Environmental DNA from seawater samples correlate with trawl catches of Subarctic, deepwater fishes

    DEFF Research Database (Denmark)

    Thomsen, Philip Francis; Møller, Peter Rask; Sigsgaard, Eva Egelyng

    2016-01-01

    depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were......Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques...... such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope...

  3. Amplification volume reduction on DNA database samples using FTA™ Classic Cards.

    Science.gov (United States)

    Wong, Hang Yee; Lim, Eng Seng Simon; Tan-Siew, Wai Fun

    2012-03-01

    The DNA forensic community always strives towards improvements in aspects such as sensitivity, robustness, and efficacy balanced with cost efficiency. Therefore our laboratory decided to study the feasibility of PCR amplification volume reduction using DNA entrapped in FTA™ Classic Card and to bring cost savings to the laboratory. There were a few concerns the laboratory needed to address. First, the kinetics of the amplification reaction could be significantly altered. Second, an increase in sensitivity might affect interpretation due to increased stochastic effects even though they were pristine samples. Third, statics might cause FTA punches to jump out of its allocated well into another thus causing sample-to-sample contamination. Fourth, the size of the punches might be too small for visual inspection. Last, there would be a limit to the extent of volume reduction due to evaporation and the possible need of re-injection of samples for capillary electrophoresis. The laboratory had successfully optimized a reduced amplification volume of 10 μL for FTA samples. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  4. Development of a Novel Self-Enclosed Sample Preparation Device for DNA/RNA Isolation in Space

    Science.gov (United States)

    Zhang, Ye; Mehta, Satish K.; Pensinger, Stuart J.; Pickering, Karen D.

    2011-01-01

    Modern biology techniques present potentials for a wide range of molecular, cellular, and biochemistry applications in space, including detection of infectious pathogens and environmental contaminations, monitoring of drug-resistant microbial and dangerous mutations, identification of new phenotypes of microbial and new life species. However, one of the major technological blockades in enabling these technologies in space is a lack of devices for sample preparation in the space environment. To overcome such an obstacle, we constructed a prototype of a DNA/RNA isolation device based on our novel designs documented in the NASA New Technology Reporting System (MSC-24811-1/3-1). This device is self-enclosed and pipette free, purposely designed for use in the absence of gravity. Our design can also be modified easily for preparing samples in space for other applications, such as flowcytometry, immunostaining, cell separation, sample purification and separation according to its size and charges, sample chemical labeling, and sample purification. The prototype of our DNA/RNA isolation device was tested for efficiencies of DNA and RNA isolation from various cell types for PCR analysis. The purity and integrity of purified DNA and RNA were determined as well. Results showed that our developed DNA/RNA isolation device offers similar efficiency and quality in comparison to the samples prepared using the standard protocol in the laboratory.

  5. Permanganate-assisted removal of PCR inhibitors during the DNA Chelex extraction from stained denim samples.

    Science.gov (United States)

    Pîrlea, Sorina; Puiu, Mihaela; Răducan, Adina; Oancea, Dumitru

    2017-03-01

    In this study, it was demonstrated that the DNA Chelex extraction combined with the permanganate assisted-oxidation is highly efficient in removing the PCR inhibitors often found in clothing materials, such as phthalocyanine. The extraction assays were conducted in saliva, blood and epithelial cells samples mixed with three oxidation-resistant dye copper(II) α-phthalocyanine, copper(II) β-phthalocyanine and tetrasulfonated copper(II) β-phthalocyanine. After DNA amplification, all samples were able to provide full DNA profiles. The permanganate/Chelex system was tested further on denim-stained samples and displayed the same ability to remove the PCR inhibitors from the commercial textile materials.

  6. Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.

    Science.gov (United States)

    Tripathi, Prashant; Moinuddin; Dixit, Kiran; Mir, Abdul Rouf; Habib, Safia; Alam, Khursheed; Ali, Asif

    2014-07-01

    Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 μM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. Quantification of periodontal pathogens in vascular, blood, and subgingival samples from patients with peripheral arterial disease or abdominal aortic aneurysms.

    Science.gov (United States)

    Figuero, Elena; Lindahl, Christeel; Marín, María José; Renvert, Stefan; Herrera, David; Ohlsson, Ola; Wetterling, Thomas; Sanz, Mariano

    2014-09-01

    The aim of this investigation is to quantify periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, and Tannerella forsythia) in vascular, blood, and subgingival samples. As a secondary objective, two molecular bacterial identification methods (nested polymerase chain reaction [PCR] and quantitative PCR [qPCR]) are compared. Seventy consecutive patients provided a vascular lesion, a blood sample, and 36 subgingival samples. Bacterial DNA was extracted, and qPCR was used to determine the prevalence and amounts of the target pathogens in each sample. Nested PCR was performed only in the samples from vascular lesions. Periodontal examination was performed in 42 patients. Mann-Whitney U or χ(2) tests were used to compare microbiologic results according to periodontal diagnosis. All targeted periodontal pathogens (A. actinomycetemcomitans, P. gingivalis, T. forsythia, or C. rectus) were detected in subgingival samples, with a prevalence rate of 72.2%, 47.2%, 74.3%, and 82.9%, respectively. In 7.1% and 11.4% of vascular and blood samples, bacterial DNA was detected. One patient was positive for A. actinomycetemcomitans in the three types of samples. No differences were found in the levels of targeted bacteria when comparing patients with and without periodontitis. Prevalence rates obtained with nested PCR were significantly higher than those obtained with qPCR. The presence of A. actinomycetemcomitans was demonstrated in vascular, blood, and subgingival samples in one of 36 patients. These results, although with a very low frequency, may support the hypothesis of a translocation of periodontal pathogens from subgingival microbiota to the bloodstream and then to atheromatous plaques in carotid or other peripheral arteries. Nested PCR is not an adequate method for identifying DNA of periodontal pathogens in low quantities because of the high number of false-negative results.

  8. Mitochondrial DNA sequence variation in Finnish patients with matrilineal diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Soini Heidi K

    2012-07-01

    Full Text Available Abstract Background The genetic background of type 2 diabetes is complex involving contribution by both nuclear and mitochondrial genes. There is an excess of maternal inheritance in patients with type 2 diabetes and, furthermore, diabetes is a common symptom in patients with mutations in mitochondrial DNA (mtDNA. Polymorphisms in mtDNA have been reported to act as risk factors in several complex diseases. Findings We examined the nucleotide variation in complete mtDNA sequences of 64 Finnish patients with matrilineal diabetes. We used conformation sensitive gel electrophoresis and sequencing to detect sequence variation. We analysed the pathogenic potential of nonsynonymous variants detected in the sequences and examined the role of the m.16189 T>C variant. Controls consisted of non-diabetic subjects ascertained in the same population. The frequency of mtDNA haplogroup V was 3-fold higher in patients with diabetes. Patients harboured many nonsynonymous mtDNA substitutions that were predicted to be possibly or probably damaging. Furthermore, a novel m.13762 T>G in MTND5 leading to p.Ser476Ala and several rare mtDNA variants were found. Haplogroup H1b harbouring m.16189 T > C and m.3010 G > A was found to be more frequent in patients with diabetes than in controls. Conclusions Mildly deleterious nonsynonymous mtDNA variants and rare population-specific haplotypes constitute genetic risk factors for maternally inherited diabetes.

  9. Ehrlichia canis morulae and DNA detection in whole blood and spleen aspiration samples.

    Science.gov (United States)

    Faria, Joice Lara Maia; Dagnone, Ana Sílvia; Munhoz, Thiago Demarchi; João, Carolina Franchi; Pereira, Wanderson Adriano Biscola; Machado, Rosângela Zacarias; Tinucci-Costa, Mirela

    2010-01-01

    The aim of this study was to compare the detection of Ehrlichia canis morulae and DNA by nPCR in whole blood and spleen aspiration. The sample included 40 dogs showing thrombocytopenia associated to clinical signs suggestive of canine ehrlichiosis. Morulae detection showed that in 35 of the dogs studied, 17 had morulae in spleen tissue, and two in buffy coat smears. E. canis DNA was detected in 29/40 blood samples. We verified that morulae detection is more efficient in cytological preparations from spleen aspiration. On the other hand, nPCR on spleen and blood samples were equally efficient for disease diagnosis.

  10. 16S rDNA-based metagenomic analysis of dental plaque and lung bacteria in patients with severe acute exacerbations of chronic obstructive pulmonary disease.

    Science.gov (United States)

    Tan, L; Wang, H; Li, C; Pan, Y

    2014-12-01

    Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are leading causes of mortality in hospital intensive care units. We sought to determine whether dental plaque biofilms might harbor pathogenic bacteria that can eventually cause lung infections in patients with severe AE-COPD. Paired samples of subgingival plaque biofilm and tracheal aspirate were collected from 53 patients with severe AE-COPD. Total bacterial DNA was extracted from each sample individually for polymerase chain reaction amplification and/or generation of bacterial 16S rDNA sequences and cDNA libraries. We used a metagenomic approach, based on bacterial 16S rDNA sequences, to compare the distribution of species present in dental plaque and lung. Analysis of 1060 sequences (20 clones per patient) revealed a wide range of aerobic, anaerobic, pathogenic, opportunistic, novel and uncultivable bacterial species. Species indistinguishable between the paired subgingival plaque and tracheal aspirate samples (97-100% similarity in 16S rDNA sequence) were dental plaque pathogens (Aggregatibacter actinomycetemcomitans, Capnocytophaga sputigena, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and lung pathogens (Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa and Streptococcus pneumoniae). Real-time polymerase chain reaction of 16S rDNA indicated lower levels of Pseudomonas aeruginosa and Porphyromonas gingivalis colonizing the dental plaques compared with the paired tracheal aspirate samples. These results support the hypothesis that dental bacteria may contribute to the pathology of severe AE-COPD. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Digital Droplet Multiple Displacement Amplification (ddMDA for Whole Genome Sequencing of Limited DNA Samples.

    Directory of Open Access Journals (Sweden)

    Minsoung Rhee

    Full Text Available Multiple displacement amplification (MDA is a widely used technique for amplification of DNA from samples containing limited amounts of DNA (e.g., uncultivable microbes or clinical samples before whole genome sequencing. Despite its advantages of high yield and fidelity, it suffers from high amplification bias and non-specific amplification when amplifying sub-nanogram of template DNA. Here, we present a microfluidic digital droplet MDA (ddMDA technique where partitioning of the template DNA into thousands of sub-nanoliter droplets, each containing a small number of DNA fragments, greatly reduces the competition among DNA fragments for primers and polymerase thereby greatly reducing amplification bias. Consequently, the ddMDA approach enabled a more uniform coverage of amplification over the entire length of the genome, with significantly lower bias and non-specific amplification than conventional MDA. For a sample containing 0.1 pg/μL of E. coli DNA (equivalent of ~3/1000 of an E. coli genome per droplet, ddMDA achieves a 65-fold increase in coverage in de novo assembly, and more than 20-fold increase in specificity (percentage of reads mapping to E. coli compared to the conventional tube MDA. ddMDA offers a powerful method useful for many applications including medical diagnostics, forensics, and environmental microbiology.

  12. Improved Methods of Carnivore Faecal Sample Preservation, DNA Extraction and Quantification for Accurate Genotyping of Wild Tigers

    Science.gov (United States)

    Harika, Katakam; Mahla, Ranjeet Singh; Shivaji, Sisinthy

    2012-01-01

    Background Non-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase. Methods We developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval. Results Average DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (pDNA degradation occurs within 3 days of exposure to direct sunlight. DNA concentrations of Day 1 samples stored by ethanol and silica methods for a month varied significantly from fresh Day 1 extracts (p0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively. Conclusions Our results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols. PMID:23071624

  13. Self-reference and random sampling approach for label-free identification of DNA composition using plasmonic nanomaterials.

    Science.gov (United States)

    Freeman, Lindsay M; Pang, Lin; Fainman, Yeshaiahu

    2018-05-09

    The analysis of DNA has led to revolutionary advancements in the fields of medical diagnostics, genomics, prenatal screening, and forensic science, with the global DNA testing market expected to reach revenues of USD 10.04 billion per year by 2020. However, the current methods for DNA analysis remain dependent on the necessity for fluorophores or conjugated proteins, leading to high costs associated with consumable materials and manual labor. Here, we demonstrate a potential label-free DNA composition detection method using surface-enhanced Raman spectroscopy (SERS) in which we identify the composition of cytosine and adenine within single strands of DNA. This approach depends on the fact that there is one phosphate backbone per nucleotide, which we use as a reference to compensate for systematic measurement variations. We utilize plasmonic nanomaterials with random Raman sampling to perform label-free detection of the nucleotide composition within DNA strands, generating a calibration curve from standard samples of DNA and demonstrating the capability of resolving the nucleotide composition. The work represents an innovative way for detection of the DNA composition within DNA strands without the necessity of attached labels, offering a highly sensitive and reproducible method that factors in random sampling to minimize error.

  14. Real-Time Polymerase Chain Reaction Detection of Angiostrongylus cantonensis DNA in Cerebrospinal Fluid from Patients with Eosinophilic Meningitis.

    Science.gov (United States)

    Qvarnstrom, Yvonne; Xayavong, Maniphet; da Silva, Ana Cristina Aramburu; Park, Sarah Y; Whelen, A Christian; Calimlim, Precilia S; Sciulli, Rebecca H; Honda, Stacey A A; Higa, Karen; Kitsutani, Paul; Chea, Nora; Heng, Seng; Johnson, Stuart; Graeff-Teixeira, Carlos; Fox, LeAnne M; da Silva, Alexandre J

    2016-01-01

    Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis. Timely diagnosis of these infections is difficult, partly because reliable laboratory diagnostic methods are unavailable. The aim of this study was to evaluate the usefulness of a real-time polymerase chain reaction (PCR) assay for the detection of A. cantonensis DNA in human cerebrospinal fluid (CSF) specimens. A total of 49 CSF specimens from 33 patients with eosinophilic meningitis were included: A. cantonensis DNA was detected in 32 CSF specimens, from 22 patients. Four patients had intermittently positive and negative real-time PCR results on subsequent samples, indicating that the level of A. cantonensis DNA present in CSF may fluctuate during the course of the illness. Immunodiagnosis and/or supplemental PCR testing supported the real-time PCR findings for 30 patients. On the basis of these observations, this real-time PCR assay can be useful to detect A. cantonensis in the CSF from patients with eosinophilic meningitis. © The American Society of Tropical Medicine and Hygiene.

  15. Comparison of three methods for recovery of Brucella canis DNA from canine blood samples.

    Science.gov (United States)

    Batinga, Maria Cryskely A; Dos Santos, Jaíne C; Lima, Julia T R; Bigotto, Maria Fernanda D; Muner, Kerstin; Faita, Thalita; Soares, Rodrigo M; da Silva, David A V; Oliveira, Trícia M F S; Ferreira, Helena L; Diniz, Jaqueline A; Keid, Lara B

    2017-12-01

    Brucella canis, a gram-negative, facultative intracellular and zoonotic bacterium causes canine brucellosis. Direct methods are the most appropriate for the detection of canine brucellosis and bacterial isolation from blood samples has been employed as gold-standard method. However, due to the delay in obtaining results and the biological risk of the bacterial culturing, the polymerase chain reaction (PCR) has been successfully used as an alternative method for the diagnosis of the infection. Sample preparation is a key step for successful PCR and protocols that provide high DNA yield and purity are recommended to ensure high diagnostic sensitivity. The objective of this study was to evaluate the performance of PCR for the diagnosis of B. canis infection in 36 dogs by testing DNA of whole blood obtained through different extraction and purification protocols. Methods 1 and 2 were based on a commercial kit, using protocols recommended for DNA purification of whole blood and tissue samples, respectively. Method 3 was an in-house method based on enzymatic lysis and purification using organic solvents. The results of the PCR on samples obtained through three different DNA extraction protocols were compared to the blood culture. Of the 36 dogs, 13 (36.1%) were positive by blood culturing, while nine (25.0%), 14 (38.8%), and 15 (41.6%) were positive by PCR after DNA extraction using methods 1, 2 and 3, respectively. PCR performed on DNA purified by Method 2 was as efficient as blood culturing and PCR performed on DNA purified with in-house method, but had the advantage of being less laborious and, therefore, a suitable alternative for the direct B. canis detection in dogs. Copyright © 2017. Published by Elsevier B.V.

  16. Direct detection of the AR-E211 G > A gene polymorphism from blood and tissue samples without DNA isolation.

    Science.gov (United States)

    Reptova, Silvie; Trtkova, Katerina Smesny; Kolar, Zdenek

    2014-04-01

    The pathogenesis of prostate cancer (CaP) involves alterations in a gene structure of the androgen receptor (AR). The single nucleotide polymorphism AR-E211 G > A localized in exon 1 of the AR gene (G1733A) was detected using direct polymerase chain reaction and restriction digestion (PCR-RFLP) method on blood and tissue samples without prior DNA isolation. We used blood samples of patients with a diagnosis of benign prostatic hyperplasia (BPH) or CaP. From monitored group of CaP patients were selected specimen in formalin-fixed paraffin-embedded tissue blocks with morphology of BPH and CaP. The main objective of our study was to develop a method based the direct PCR-RFLP analysis from blood and tissue without prior DNA isolation for faster genotyping analysis of a large number of samples. We found no statistically significant differences in allelic % of the AR-E211 G > A polymorphism between BPH and CaP patients (p ≤ 0.8462). Genotyping of the AR-E211 G > A variant in blood was not identical with tumor tissue genotyping analysis. Significant agreement between blood and tissue AR-E211 G > A polymorphism only in non-tumor tissue focus was confirmed. Although we analyzed a limited number of the tissue samples, we suppose that a presence of the minor allele A may be associated with cancer transformation-induced changes of the modified AR gene.

  17. cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

    Science.gov (United States)

    Antunes, Heliton S; Wajnberg, Gabriel; Pinho, Marcos B; Jorge, Natasha Andressa Nogueira; de Moraes, Joyce Luana Melo; Stefanoff, Claudio Gustavo; Herchenhorn, Daniel; Araújo, Carlos M M; Viégas, Celia Maria Pais; Rampini, Mariana P; Dias, Fernando L; de Araujo-Souza, Patricia Savio; Passetti, Fabio; Ferreira, Carlos G

    2018-01-01

    Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

  18. Impact of Somatic Mutations in the D-Loop of Mitochondrial DNA on the Survival of Oral Squamous Cell Carcinoma Patients

    Science.gov (United States)

    Lin, Jin-Ching; Wang, Chen-Chi; Jiang, Rong-San; Wang, Wen-Yi; Liu, Shih-An

    2015-01-01

    Objectives The aim of this study was to investigate somatic mutations in the D-loop of mitochondrial DNA (mtDNA) and their impact on survival in oral squamous cell carcinoma patients. Materials and Methods Surgical specimen confirmed by pathological examination and corresponding non-cancerous tissues were collected from 120 oral squamous cell carcinoma patients. The sequence in the D-loop of mtDNA from non-cancerous tissues was compared with that from paired cancer samples and any sequence differences were recognized as somatic mutations. Results Somatic mutations in the D-loop of mtDNA were identified in 75 (62.5%) oral squamous cell carcinoma patients and most of them occurred in the poly-C tract. Although there were no significant differences in demographic and tumor-related features between participants with and without somatic mutation, the mutation group had a better survival rate (5 year disease-specific survival rate: 64.0% vs. 43.0%, P = 0.0266). Conclusion Somatic mutation in D-loop of mtDNA was associated with a better survival in oral squamous cell carcinoma patients. PMID:25906372

  19. DNA methylation changes separate allergic patients from healthy controls and may reflect altered CD4+ T-cell population structure.

    Directory of Open Access Journals (Sweden)

    Colm E Nestor

    2014-01-01

    Full Text Available Altered DNA methylation patterns in CD4(+ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (N(patients = 8, N(controls = 8 and gene expression (N(patients = 9, Ncontrols = 10 profiles of CD4(+ T-cells from SAR patients and healthy controls using Illumina's HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (N(patients = 12, N(controls = 12, but not by gene expression (N(patients = 21, N(controls = 21 was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (N(patients = 35 and controls (N(controls = 12, which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4(+ T cells.

  20. Evaluation of five DNA extraction methods for purification of DNA from atherosclerotic tissue and estimation of prevalence of Chlamydia pneumoniae in tissue from a Danish population undergoing vascular repair

    Directory of Open Access Journals (Sweden)

    Lindholt Jes S

    2003-09-01

    Full Text Available Abstract Background To date PCR detection of Chlamydia pneumoniae DNA in atherosclerotic lesions from Danish patients has been unsuccessful. To establish whether non-detection was caused by a suboptimal DNA extraction method, we tested five different DNA extraction methods for purification of DNA from atherosclerotic tissue. Results The five different DNA extraction methods were tested on homogenate of atherosclerotic tissue spiked with C. pneumoniae DNA or EB, on pure C. pneumoniae DNA samples and on whole C. pneumoniae EB. Recovery of DNA was measured with a C. pneumoniae-specific quantitative real-time PCR. A DNA extraction method based on DNA-binding to spin columns with a silica-gel membrane (DNeasy Tissue kit showed the highest recovery rate for the tissue samples and pure DNA samples. However, an automated extraction method based on magnetic glass particles (MagNA Pure performed best on intact EB and atherosclerotic tissue spiked with EB. The DNeasy Tissue kit and MagNA Pure methods and the highly sensitive real-time PCR were subsequently used on 78 atherosclerotic tissue samples from Danish patients undergoing vascular repair. None of the samples were positive for C. pneumoniae DNA. The atherosclerotic samples were tested for inhibition by spiking with two different, known amounts of C. pneumoniae DNA and no samples showed inhibition. Conclusion As a highly sensitive PCR method and an optimised DNA extraction method were used, non-detection in atherosclerotic tissue from the Danish population was probably not caused by use of inappropriate methods. However, more samples may need to be analysed per patient to be completely certain on this. Possible methodological and epidemiological reasons for non-detection of C. pneumoniae DNA in atherosclerotic tissue from the Danish population are discussed. Further testing of DNA extraction methods is needed as this study has shown considerable intra- and inter-method variation in DNA recovery.

  1. [Real-time quantification to analyze historical Colombian samples detecting a short fragment of hypervariable region II of mitochondrial DNA].

    Science.gov (United States)

    Pérez, Luz Adriana; Rodríguez, Freddy; Langebaek, Carl Henrik; Groot, Helena

    2016-09-01

    Unlike other molecular biology studies, the analysis of ancient DNA (aDNA) requires special infrastructure and methodological conditions to guarantee the quality of the results. One of the main authenticity criteria is DNA quantification, where quantitative real-time PCR is often used given its sensitivity and specificity. Nevertheless, the implementation of these conditions and methodologies to fulfill authenticity criteria imply higher costs. Objective: To develop a simple and less costly method for mitochondrial DNA quantification suitable for highly degraded samples. Materials and methods: The proposed method is based on the use of mini-primers for the specific amplification of short fragments of mitochondrial DNA. The subsequent purification of these amplified fragments allows a standard curve to be constructed with concentrations in accordance to the state of degradation of the samples. Results: The proposed method successfully detected DNA from ancient samples including bone remains and mummified tissue. DNA inhibitory substances were also detected. Conclusion: The proposed method represents a simpler and cost-effective way to detect low amounts of aDNA, and a tool to differentiate DNA-free samples from samples with inhibitory substances.

  2. Radioresistant DNA synthesis in fibroblasts of a patient with Down's syndrome

    International Nuclear Information System (INIS)

    Barenfel'd, L.S.; Bil'din, V.N.; Pleskach, N.M.; Prokof'eva, V.V.

    1985-01-01

    Ionizing radiation effect on DNA replication on fibroblasts of a healthy donor and a patient with Down's syndrome either by direct 3 H-thymidine inclusion into DNA, or by analysis of the sizes of daughter DNA moleculs at the state of stable distribution in acid saccharose, gradients was studied. Gamma-radiation doses (5-10 Gy) suppressing DNA synthesis in normal fibroblasts practically had no effect on DNA synthesisin fibroblasts of a patient with Down's syndrome. Radioresistant DNA synthesis in Down's syndrome is conditioned by a far less supression of replicon initiation as compared with the one in normal cells. So, it is stated that in Down's disease there is no delay in DNA synthesis by ionizing radiation that enables the normal cells to repair DNA damages before replication renewal

  3. Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.

    Directory of Open Access Journals (Sweden)

    Gemma Henderson

    Full Text Available Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However

  4. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    International Nuclear Information System (INIS)

    Jackson, Christopher B.; Gallati, Sabina; Schaller, André

    2012-01-01

    Highlights: ► Serial qPCR accurately determines fragmentation state of any given DNA sample. ► Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. ► Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. ► Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze–thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA (λ nDNA ) and mtDNA (λ mtDNA ) we present an approach to possibly correct measurements in degraded samples in the future. To our knowledge this is the first time different degradation impact of the two

  5. qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serial qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct measurements in

  6. Quantitative analysis of plasma cell-free DNA and its DNA integrity in patients with metastatic prostate cancer using ALU sequence

    International Nuclear Information System (INIS)

    Fawzy, A.; Sweify, K.M.; Nofal, N.; El-Fayoumy, H.M.

    2016-01-01

    Background: Prostate cancer (PC) is the most common cancer affecting men, it accounts for 29% of all male cancer and 11% of all male cancer related death. DNA is normally released from an apoptotic source which generates small fragments of cell-free DNA, whereas cancer patients have cell-free circulating DNA that originated from necrosis, autophagy, or mitotic catastrophe, which produce large fragments. Aim of work: Differentiate the cell free DNA levels (cfDNA) and its integrity in prostate cancer patients and control group composed of benign prostate hyperplasia (BPH) and healthy persons. Methodology: cf-DNA levels were quantified by real-time PCR amplification in prostate cancer patients ( n = 50), (BPH) benign prostate hyperplasia ( n = 25) and healthy controls ( n = 30) using two sets of ALU gene (product size of 115 bp and 247-bp) and its integrity was calculated as a ratio of qPCR results of 247 bp ALU over 115 bp ALU. Results: Highly significant levels of cf-DNA and its integrity in PC patients compared to BPH. Twenty-eight (56%) patients with prostate cancer had bone metastasis. ALU115 qpcr is superior to the other markers in discriminating metastatic patients with a sensitivity of 96.4% and a specificity of 86.4% and (AUC = 0.981) Conclusion: ALU115 qpcr could be used as a valuable biomarker helping in identifying high risk patients, indicating early spread of tumor cells as a potential seed for future metastases

  7. Metabolic rescue in pluripotent cells from patients with mtDNA disease.

    Science.gov (United States)

    Ma, Hong; Folmes, Clifford D L; Wu, Jun; Morey, Robert; Mora-Castilla, Sergio; Ocampo, Alejandro; Ma, Li; Poulton, Joanna; Wang, Xinjian; Ahmed, Riffat; Kang, Eunju; Lee, Yeonmi; Hayama, Tomonari; Li, Ying; Van Dyken, Crystal; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Koski, Amy; Mitalipov, Nargiz; Amato, Paula; Wolf, Don P; Huang, Taosheng; Terzic, Andre; Laurent, Louise C; Izpisua Belmonte, Juan Carlos; Mitalipov, Shoukhrat

    2015-08-13

    Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

  8. DNA extraction in Echinococcus granulosus and Taenia spp. eggs in dogs stool samples applying thermal shock.

    Science.gov (United States)

    Hidalgo, Alejandro; Melo, Angélica; Romero, Fernando; Hidalgo, Víctor; Villanueva, José; Fonseca-Salamanca, Flery

    2018-03-01

    The extraction of DNA in taeniid eggs shows complications attached to the composition of stool samples and the high resistance of eggs to degradation. The objective of this study was to test a method of DNA extraction in taeniid eggs by applying a thermal shock to facilitate the chemical-enzymatic degradation of these elements. A group of six tubes containing 1 ml of dog stool sample was spiked with eggs of Echinococcus granulosus and another group of six with Taenia pisiformis. Samples were floated with supersaturated sugar solution and centrifuged. The upper portion of each tube (500 μl) was aspirated and deposited in 1.5 ml tubes. Three tubes from each group were incubated at -20 °C and then at 90 °C, the remaining three from each group, incubated at room temperature. Proteinase K and lysis buffer were added to each tube and incubated for 12 h at 58 °C. The lysis effect was evaluated by microscopy at 3, 6 and 12 h and integrity by electrophoresis in 1% agarose gels. With the same experimental scheme, the thermal shock effect was evaluated in extractions of 1, 2, 3 and 4 eggs of each species and the DNA was quantified. Additionally, the protocol was applied in samples of 4 dogs diagnosed with natural infection by Taeniidae worms. Finally, all the extractions were tested by PCR amplification. Both E. granulosus and T. pisiformis eggs showed a similar response in the tests. In samples without treatment, the lysis effect was poor and showed no differences over time, but in those subjected to thermal shock, eggs degradation increased with time. In both treatments, there was no DNA loss integrity. The protocol applied to limited amounts of eggs yielded PCR products in 100% of the samples exposed to thermal shock, allowing PCR amplifications up to 1 egg. In non-exposed samples, the results were not replicable. However, DNA quantification showed low values in both treatments. In turn, DNA extractions with thermal shock in infected dog samples

  9. Determination of cDNA encoding BCR/ABL fusion gene in patients with chronic myelogenous leukemia using a novel FRET-based quantum dots-DNA nanosensor.

    Science.gov (United States)

    Shamsipur, Mojtaba; Nasirian, Vahid; Barati, Ali; Mansouri, Kamran; Vaisi-Raygani, Asad; Kashanian, Soheila

    2017-05-08

    In the present study, we developed a sensitive method based on fluorescence resonance energy transfer (FRET) for the determination of the BCR/ABL fusion gene, which is used as a biomarker to confirm the clinical diagnosis of both chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). For this purpose, CdTe quantum dots (QDs) were conjugated to amino-modified 18-mer oligonucleotide ((N)DNA) to form the QDs-(N)DNA nanosensor. In the presence of methylene blue (MB) as an intercalator, the hybridization of QDs-(N)DNA with the target BCR/ABL fusion gene (complementary DNA), brings the MB (acceptor) at close proximity of the QDs (donor), leading to FRET upon photoexcitation of the QDs. The enhancement in the emission intensity of MB was used to follow up the hybridization, which was linearly proportional to concentration of the target complementary DNA in a range from 1.0 × 10 -9 to 1.25 × 10 -7  M. The detection limit of the proposed method was obtained to be 1.5 × 10 -10  M. Finally, the feasibility and selectivity of the proposed nanosensor was evaluated by the analysis of derived nucleotides from both mismatched sequences and clinical samples of patients with leukemia as real samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

    Science.gov (United States)

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

    2014-01-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

  11. Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.

    Science.gov (United States)

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H C; De Keersmaecker, Sigrid C J

    2014-12-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at -20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.

  12. Self-sampling for human papillomavirus DNA detection: a preliminary study of compliance and feasibility in BOLIVIA.

    Science.gov (United States)

    Surriabre, Pedro; Allende, Gustavo; Prado, Marcela; Cáceres, Leyddy; Bellot, Diego; Torrico, Andrea; Ustariz, Karina; Rojas, Shirley; Barriga, Jaime; Calle, Pamela; Villarroel, Ligia; Yañez, Rosse Mary; Baay, Marc; Rodriguez, Patricia; Fontaine, Véronique

    2017-12-22

    Cervical cancer incidence and mortality rates in Bolivia are among the highest in Latin America. This investigation aims to evaluate the possibility of using simple devices, e.g. a cotton swab and a glass slide, for self-sampling in order to detect human papillomavirus (HPV) DNA by PCR in cervico-vaginal cells. In the first phase of our study we evaluated the use of a glass slide as a transport medium for cervical cells. A physician took paired-cervical samples from 235 women. One sample was transported in Easyfix® solution and the other sample was smeared over a glass slide. Both were further analyzed and compared for human DNA recovery and HPV detection. A kappa value was determined to evaluate the agreement between the HPV DNA detection rates. In the second phase of the study, 222 women from the urban, peri-urban and rural regions of Cochabamba were requested to perform self-sampling using the following devices: a cotton swab combined with a glass slide, and a vaginal tampon. Women gave their opinion about the self-sampling technique. Finally, the agreement for high risk-HPV detection between self- and physician-collected samples was performed in 201 samples in order to evaluate the self-sampling technique. Firstly, the comparison between Easyfix® solution and the glass slide to transport clinical samples gave a good agreement for HPV DNA detection (κ = 0.71, 95% CI 0.60-0.81). Secondly, self-sampling, especially with cotton swab combined with glass slide, would generally be preferred over clinician sampling for a screening program based on HPV detection. Finally, we showed a good agreement between self- and physician collected samples for high risk-HPV detection (κ = 0.71, 95% CI 0.55-0.88). Simple devices such as a cotton swab and a glass slide can be used to perform self-sampling and HPV DNA detection. Furthermore, most Bolivian women preferred self-sampling over clinician-sampling for cervical cancer screening.

  13. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Directory of Open Access Journals (Sweden)

    Žel Jana

    2006-08-01

    Full Text Available Abstract Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was

  14. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Science.gov (United States)

    Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

    2006-01-01

    Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

  15. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples.

    Science.gov (United States)

    Miller, D N; Bryant, J E; Madsen, E L; Ghiorse, W C

    1999-11-01

    We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed

  16. Lymphocyte DNA damage and oxidative stress in patients with iron deficiency anemia.

    Science.gov (United States)

    Aslan, Mehmet; Horoz, Mehmet; Kocyigit, Abdurrahim; Ozgonül, Saadet; Celik, Hakim; Celik, Metin; Erel, Ozcan

    2006-10-10

    Oxidant stress has been shown to play an important role in the pathogenesis of iron deficiency anemia. The aim of this study was to investigate the association between lymphocyte DNA damage, total antioxidant capacity and the degree of anemia in patients with iron deficiency anemia. Twenty-two female with iron deficiency anemia and 22 healthy females were enrolled in the study. Peripheral DNA damage was assessed using alkaline comet assay and plasma total antioxidant capacity was determined using an automated measurement method. Lymphocyte DNA damage of patients with iron deficiency anemia was significantly higher than controls (ptotal antioxidant capacity was significantly lower (ptotal antioxidant capacity and hemoglobin levels (r=0.706, ptotal antioxidant capacity and hemoglobin levels were negatively correlated with DNA damage (r=-0.330, p<0.05 and r=-0.323, p<0.05, respectively). In conclusion, both oxidative stress and DNA damage are increased in IDA patients. Increased oxidative stress seems as an important factor that inducing DNA damage in those IDA patients. The relationships of oxidative stress and DNA damage with the severity of anemia suggest that both oxidative stress and DNA damage may, in part, have a role in the pathogenesis of IDA.

  17. Quantification of cell-free DNA in blood plasma and DNA damage degree in lymphocytes to evaluate dysregulation of apoptosis in schizophrenia patients.

    Science.gov (United States)

    Ershova, E S; Jestkova, E M; Chestkov, I V; Porokhovnik, L N; Izevskaya, V L; Kutsev, S I; Veiko, N N; Shmarina, G; Dolgikh, O; Kostyuk, S V

    2017-04-01

    Oxidative DNA damage has been proposed as one of the causes of schizophrenia (SZ), and post mortem data indicate a dysregulation of apoptosis in SZ patients. To evaluate apoptosis in vivo we quantified the concentration of plasma cell-free DNA (cfDNA index, determined using fluorescence), the levels of 8-oxodG in cfDNA (immunoassay) and lymphocytes (FL1-8-oxodG index, flow cytometry) of male patients with acute psychotic disorders: paranoid SZ (total N = 58), schizophreniform (N = 11) and alcohol-induced (N = 14) psychotic disorder, and 30 healthy males. CfDNA in SZ (N = 58) does not change compared with controls. In SZ patients. Elevated levels of 8-oxodG were found in cfDNA (N = 58) and lymphocytes (n = 45). The main sources of cfDNA are dying cells with oxidized DNA. Thus, the cfDNA/FL1-8-oxodG ratio shows the level of apoptosis in damaged cells. Two subgroups were identified among the SZ patients (n = 45). For SZ-1 (31%) and SZ-2 (69%) median values of cfDNA/FL1-8-oxodG index are related as 1:6 (p DNA in the patient's body tissues and may be a contributing cause of acute psychotic disorder. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Analysis of multiple single nucleotide polymorphisms (SNP) on DNA traces from plasma and dried blood samples

    NARCIS (Netherlands)

    Catsburg, Arnold; van der Zwet, Wil C.; Morre, Servaas A.; Ouburg, Sander; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2007-01-01

    Reliable analysis of single nucleotide polymorphisms (SNPs) in DNA derived from samples containing low numbers of cells or from suboptimal sources can be difficult. A new procedure to characterize multiple SNPs in traces of DNA from plasma and old dried blood samples was developed. Six SNPs in the

  19. Prevalence and persistence of male DNA identified in mixed saliva samples after intense kissing.

    Science.gov (United States)

    Kamodyová, Natália; Durdiaková, Jaroslava; Celec, Peter; Sedláčková, Tatiana; Repiská, Gabriela; Sviežená, Barbara; Minárik, Gabriel

    2013-01-01

    Identification of foreign biological material by genetic profiling is widely used in forensic DNA testing in different cases of sexual violence, sexual abuse or sexual harassment. In all these kinds of sexual assaults, the perpetrator could constrain the victim to kissing. The value of the victim's saliva taken after such an assault has not been investigated in the past with currently widely used molecular methods of extremely high sensitivity (e.g. qPCR) and specificity (e.g. multiplex Y-STR PCR). In our study, 12 voluntary pairs were tested at various intervals after intense kissing and saliva samples were taken from the women to assess the presence of male DNA. Sensitivity-focused assays based on the SRY (single-copy gene) and DYS (multi-copy gene) sequence motifs confirmed the presence of male DNA in female saliva after 10 and even 60min after kissing, respectively. For specificity, standard multiplex Y-STR PCR profiling was performed and male DNA was found in female saliva samples, as the entire Y-STR profile, even after 30min in one sample. Our study confirms that foreign DNA tends to persist for a restricted period of time in the victim's mouth, can be isolated from saliva after prompt collection and can be used as a valuable source of evidence. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  20. Bacteria and bacterial DNA in atherosclerotic plaque and aneurysmal wall biopsies from patients with and without periodontitis

    Directory of Open Access Journals (Sweden)

    Zahra Armingohar

    2014-05-01

    Full Text Available Background: Several studies have reported an association between chronic periodontitis (CP and cardiovascular diseases. Detection of periodontopathogens, including red complex bacteria (RCB, in vascular lesions has suggested these bacteria to be involved in the pathogenesis of atherosclerosis and abdominal aortic aneurysms. Objective: In this study, we investigate bacteria and their DNA in vascular biopsies from patients with vascular diseases (VD; i.e. abdominal aortic aneurysms, atherosclerotic carotid, and common femoral arteries, with and without CP. Methods: DNA was extracted from vascular biopsies selected from 40 VD patients: 30 with CP and 10 without CP. The V3-V5 region of the 16S rDNA (V3-V5 was polymerase chain reaction (PCR-amplified, and the amplicons were cloned into Escherichia coli, sequenced, and classified (GenBank and the Human Oral Microbiome database. Species-specific primers were used for the detection of Porphyromonas gingivalis. In addition, 10 randomly selected vascular biopsies from the CP group were subjected to scanning electron microscopy (SEM for visualization of bacteria. Checkerboard DNA–DNA hybridization was performed to assess the presence of RCB in 10 randomly selected subgingival plaque samples from CP patients. Results: A higher load and mean diversity of bacteria were detected in vascular biopsies from VD patients with CP compared to those without CP. Enterobacteriaceae were frequently detected in vascular biopsies together with cultivable, commensal oral, and not-yet-cultured bacterial species. While 70% of the subgingival plaque samples from CP patients showed presence of RCB, only P. gingivalis was detected in one vascular biopsy. Bacterial cells were seen in all 10 vascular biopsies examined by SEM. Conclusions: A higher bacterial load and more diverse colonization were detected in VD lesions of CP patients as compared to patients without CP. This indicated that a multitude of bacterial species both

  1. Pseudogenes and DNA-based diet analyses: A cautionary tale from a relatively well sampled predator-prey system

    DEFF Research Database (Denmark)

    Dunshea, G.; Barros, N. B.; Wells, R. S.

    2008-01-01

    Mitochondrial ribosomal DNA is commonly used in DNA-based dietary analyses. In such studies, these sequences are generally assumed to be the only version present in DNA of the organism of interest. However, nuclear pseudogenes that display variable similarity to the mitochondrial versions...... are common in many taxa. The presence of nuclear pseudogenes that co-amplify with their mitochondrial paralogues can lead to several possible confounding interpretations when applied to estimating animal diet. Here, we investigate the occurrence of nuclear pseudogenes in fecal samples taken from bottlenose...... dolphins (Tursiops truncatus) that were assayed for prey DNA with a universal primer technique. We found pseudogenes in 13 of 15 samples and 1-5 pseudogene haplotypes per sample representing 5-100% of all amplicons produced. The proportion of amplicons that were pseudogenes and the diversity of prey DNA...

  2. Establishing a novel automated magnetic bead-based method for the extraction of DNA from a variety of forensic samples.

    Science.gov (United States)

    Witt, Sebastian; Neumann, Jan; Zierdt, Holger; Gébel, Gabriella; Röscheisen, Christiane

    2012-09-01

    Automated systems have been increasingly utilized for DNA extraction by many forensic laboratories to handle growing numbers of forensic casework samples while minimizing the risk of human errors and assuring high reproducibility. The step towards automation however is not easy: The automated extraction method has to be very versatile to reliably prepare high yields of pure genomic DNA from a broad variety of sample types on different carrier materials. To prevent possible cross-contamination of samples or the loss of DNA, the components of the kit have to be designed in a way that allows for the automated handling of the samples with no manual intervention necessary. DNA extraction using paramagnetic particles coated with a DNA-binding surface is predestined for an automated approach. For this study, we tested different DNA extraction kits using DNA-binding paramagnetic particles with regard to DNA yield and handling by a Freedom EVO(®)150 extraction robot (Tecan) equipped with a Te-MagS magnetic separator. Among others, the extraction kits tested were the ChargeSwitch(®)Forensic DNA Purification Kit (Invitrogen), the PrepFiler™Automated Forensic DNA Extraction Kit (Applied Biosystems) and NucleoMag™96 Trace (Macherey-Nagel). After an extensive test phase, we established a novel magnetic bead extraction method based upon the NucleoMag™ extraction kit (Macherey-Nagel). The new method is readily automatable and produces high yields of DNA from different sample types (blood, saliva, sperm, contact stains) on various substrates (filter paper, swabs, cigarette butts) with no evidence of a loss of magnetic beads or sample cross-contamination. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Analysis of fingerprint samples, testing various conditions, for forensic DNA identification.

    Science.gov (United States)

    Ostojic, Lana; Wurmbach, Elisa

    2017-01-01

    Fingerprints can be of tremendous value for forensic biology, since they can be collected from a wide variety of evident types, such as handles of weapons, tools collected in criminal cases, and objects with no apparent staining. DNA obtained from fingerprints varies greatly in quality and quantity, which ultimately affects the quality of the resulting STR profiles. Additional difficulties can arise when fingerprint samples show mixed STR profiles due to the handling of multiple persons. After applying a tested protocol for sample collection (swabbing with 5% Triton X-100), DNA extraction (using an enzyme that works at elevated temperatures), and PCR amplification (AmpFlSTR® Identifiler® using 31cycles) extensive analysis was performed to better understand the challenges inherent to fingerprint samples, with the ultimate goal of developing valuable profiles (≥50% complete). The impact of time on deposited fingerprints was investigated, revealing that while the quality of profiles deteriorated, full STR profiles could still be obtained from samples after 40days of storage at room temperature. By comparing the STR profiles from fingerprints of the dominant versus the non-dominant hand, we found a slightly better quality from the non-dominant hand, which was not always significant. Substrates seem to have greater effects on fingerprints. Tests on glass, plastic, paper and metal (US Quarter dollar, made of Cu and Ni), common substrates in offices and homes, showed best results for glass, followed by plastic and paper, while almost no profiles were obtained from a Quarter dollar. Important for forensic casework, we also assessed three-person mixtures of touched fingerprint samples. Unlike routinely used approaches for sampling evidence, the surface of an object (bottle) was sectioned into six equal parts and separate samples were taken from each section. The samples were processed separately for DNA extraction and STR amplification. The results included a few single

  4. Detection of Babesia annae DNA in lung exudate samples from Red foxes (Vulpes vulpes) in Great Britain.

    Science.gov (United States)

    Bartley, Paul M; Hamilton, Clare; Wilson, Cari; Innes, Elisabeth A; Katzer, Frank

    2016-02-12

    This study aimed to determine the prevalence of Babesia species DNA in lung exudate samples collected from red foxes (Vulpes vulpes) from across Great Britain. Babesia are small piroplasmid parasites which are mainly transmitted through the bite of infected ticks of the family Ixodidae. Babesia can cause potentially fatal disease in a wide-range of mammalian species including humans, dogs and cattle, making them of significant economic importance to both the medical and veterinary fields. DNA was extracted from lung exudate samples of 316 foxes. A semi-nested PCR was used to initially screen samples, using universal Babesia-Theileria primers which target the 18S rRNA gene. A selection of positive PCR amplicons were purified and sequenced. Subsequently specific primers were designed to detect Babesia annae and used to screen all 316 DNA samples. Randomly selected positive samples were purified and sequenced (GenBank accession KT580786). Clones spanning a 1717 bp region of the 18S rRNA gene were generated from 2 positive samples, the resultant consensus sequence was submitted to GenBank (KT580785). Sequence KT580785 was used in the phylogenetic analysis Babesia annae DNA was detected in the fox samples, in total 46/316 (14.6%) of samples tested positive for the presence of Babesia annae DNA. The central region of England had the highest prevalence at 36.7%, while no positive samples were found from Wales, though only 12 samples were tested from this region. Male foxes were found to have a higher prevalence of Babesia annae DNA than females in all regions of Britain. Phylogenetic and sequence analysis of the GenBank submissions (Accession numbers KT580785 and KT580786) showed 100% identity to Babesia sp.-'Spanish Dog' (AY534602, EU583387 and AF188001). This is the first time that Babesia annae DNA has been reported in red foxes in Great Britain with positive samples being found across England and Scotland indicating that this parasite is well established within the

  5. An economical mtDNA SNP assay detecting different mitochondrial haplogroups in identical HVR 1 samples of Caucasian ancestry.

    Science.gov (United States)

    Köhnemann, Stephan; Hohoff, Carsten; Pfeiffer, Heidi

    2009-09-01

    We had sequenced 329 Caucasian samples in Hypervariable Region 1 (HVR 1) and found that they belong to eleven different mitochondrial DNA (mtDNA) haplotypes. The sample set was further analysed by an mtDNA assay examining 32 single nucleotide polymorphisms (SNPs) for haplogroup discrimination. In a validation study on 160 samples of different origin it was shown that these SNPs were able to discriminate between the evolved superhaplogroups worldwide (L, M and N) and between the nine most common Caucasian haplogroups (H, I, J, K, T, U, V, W and X). The 32 mtDNA SNPs comprised 42 different SNP haplotypes instead of only eleven haplotypes after HVR 1 sequencing. The assay provided stable results in a range of 5ng genomic DNA down to virtually no genomic DNA per reaction. It was possible to detect samples of African, Asian and Eurasian ancestry, respectively. The 32 mtDNA SNP assay is a helpful adjunct to further distinguish between identical HVR 1 sequences of Caucasian origin. Our results suggest that haplogroup prediction using HVR 1 sequencing provides instable results. The use of coding region SNPs for haplogroup assignment is more suited than using HVR 1 haplotypes.

  6. Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

    International Nuclear Information System (INIS)

    Izui, S.; Lambert, P.H.; Miescher, P.A.

    1977-01-01

    The presence of DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of [ 3 H]actinomycin D ([ 3 H]ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in SLE sera was very low. Only 6% of sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the [ 3 H]ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE. (author)

  7. Potentialities of aberrantly methylated circulating DNA for diagnostics and post-treatment follow-up of lung cancer patients.

    Science.gov (United States)

    Ponomaryova, Anastasia A; Rykova, Elena Yu; Cherdyntseva, Nadezda V; Skvortsova, Tatiana E; Dobrodeev, Alexey Yu; Zav'yalov, Alexander A; Bryzgalov, Leonid O; Tuzikov, Sergey A; Vlassov, Valentin V; Laktionov, Pavel P

    2013-09-01

    To date, aberrant DNA methylation has been shown to be one of the most common and early causes of malignant cell transformation and tumors of different localizations, including lung cancer. Cancer cell-specific methylated DNA has been found in the blood of cancer patients, indicating that cell-free DNA circulating in the blood (cirDNA) is a convenient tumor-associated DNA marker that can be used as a minimally invasive diagnostic test. In the current study, we investigated the methylation status in blood samples of 32 healthy donors and 60 lung cancer patients before and after treatment with neoadjuvant chemotherapy followed by total tumor resection. Using quantitative methylation-specific PCR, we found that the index of methylation (IM), calculated as IM = 100 × [copy number of methylated/(copy number of methylated + unmethylated gene)], for the RASSF1A and RARB2 genes in the cirDNA isolated from blood plasma and cell-surface-bound cirDNA was elevated 2- to 3-fold in lung cancer patients compared with healthy donors. Random forest classification tree model based on these variables combined (RARB2 and RASSF1A IM in both plasma and cell-surface-bound cirDNA) lead to NSCLC patients' and healthy subjects' differentiation with 87% sensitivity and 75% specificity. An association of increased IM values with an advanced stage of non-small-cell lung cancer was found for RARB2 but not for RASSF1A. Chemotherapy and total tumor resection resulted in a significant decrease in the IM for RARB2 and RASSF1A, in both cirDNA fractions, comparable to the IM level of healthy subjects. Importantly, a rise in the IM for RARB2 was detected in patients within the follow-up period, which manifested in disease relapse at 9 months, confirmed with instrumental and pathologic methods. Our data indicate that quantitative analysis of the methylation status of the RARB2 and RASSF1A tumor suppressor genes in both cirDNA fractions is a useful tool for lung cancer diagnostics, evaluation of cancer

  8. Evaluating sampling strategy for DNA barcoding study of coastal and inland halo-tolerant Poaceae and Chenopodiaceae: A case study for increased sample size.

    Directory of Open Access Journals (Sweden)

    Peng-Cheng Yao

    Full Text Available Environmental conditions in coastal salt marsh habitats have led to the development of specialist genetic adaptations. We evaluated six DNA barcode loci of the 53 species of Poaceae and 15 species of Chenopodiaceae from China's coastal salt marsh area and inland area. Our results indicate that the optimum DNA barcode was ITS for coastal salt-tolerant Poaceae and matK for the Chenopodiaceae. Sampling strategies for ten common species of Poaceae and Chenopodiaceae were analyzed according to optimum barcode. We found that by increasing the number of samples collected from the coastal salt marsh area on the basis of inland samples, the number of haplotypes of Arundinella hirta, Digitaria ciliaris, Eleusine indica, Imperata cylindrica, Setaria viridis, and Chenopodium glaucum increased, with a principal coordinate plot clearly showing increased distribution points. The results of a Mann-Whitney test showed that for Digitaria ciliaris, Eleusine indica, Imperata cylindrica, and Setaria viridis, the distribution of intraspecific genetic distances was significantly different when samples from the coastal salt marsh area were included (P < 0.01. These results suggest that increasing the sample size in specialist habitats can improve measurements of intraspecific genetic diversity, and will have a positive effect on the application of the DNA barcodes in widely distributed species. The results of random sampling showed that when sample size reached 11 for Chloris virgata, Chenopodium glaucum, and Dysphania ambrosioides, 13 for Setaria viridis, and 15 for Eleusine indica, Imperata cylindrica and Chenopodium album, average intraspecific distance tended to reach stability. These results indicate that the sample size for DNA barcode of globally distributed species should be increased to 11-15.

  9. Usage of DNA Fingerprinting Technology for Quality Control in Molecular Lab Bench Work.

    Science.gov (United States)

    McIntosh, Linda Y; Lal, Janella E; Qin, Dahui

    2018-01-01

    One of the major quality assurance (QA) goals in many molecular laboratories is to avoid sample pipetting errors on the lab bench; especially when pipetting into multiwell plates. A pipetting error can cause a switch in patient samples, which can lead to recording the wrong results for the patient samples involved. Such pipetting errors are difficult to identify when it happens in lab bench work. DNA fingerprinting is a powerful tool in determining sample identities. Our laboratory has explored the usage of this technology in our QA process and successfully established that DNA fingerprinting can be used to monitor possible sample switch in gene rearrangement lab bench work. We use florescent light to quench the florescence in the gene rearrangement polymerase chain reaction products. After that, DNA fingerprinting technology is used to identify the sample DNA in the gene rearrangement polymerase chain reaction plate. The result is compared with the corresponding patient's blood sample DNA to determine whether there is a sample switch during the lab bench work.

  10. Comparative evaluation of PCR amplification of RLEP, 16S rRNA, rpoT and Sod A gene targets for detection of M. leprae DNA from clinical and environmental samples.

    Science.gov (United States)

    Turankar, Ravindra P; Pandey, Shradha; Lavania, Mallika; Singh, Itu; Nigam, Astha; Darlong, Joydeepa; Darlong, Fam; Sengupta, Utpal

    2015-03-01

    PCR assay is a highly sensitive, specific and reliable diagnostic tool for the identification of pathogens in many infectious diseases. Genome sequencing Mycobacterium leprae revealed several gene targets that could be used for the detection of DNA from clinical and environmental samples. The PCR sensitivity of particular gene targets for specific clinical and environmental isolates has not yet been established. The present study was conducted to compare the sensitivity of RLEP, rpoT, Sod A and 16S rRNA gene targets in the detection of M. leprae in slit skin smear (SSS), blood, soil samples of leprosy patients and their surroundings. Leprosy patients were classified into Paucibacillary (PB) and Multibacillary (MB) types. Ziehl-Neelsen (ZN) staining method for all the SSS samples and Bacteriological Index (BI) was calculated for all patients. Standard laboratory protocol was used for DNA extraction from SSS, blood and soil samples. PCR technique was performed for the detection of M. leprae DNA from all the above-mentioned samples. RLEP gene target was able to detect the presence of M. leprae in 83% of SSS, 100% of blood samples and in 36% of soil samples and was noted to be the best out of all other gene targets (rpoT, Sod A and 16S rRNA). It was noted that the RLEP gene target was able to detect the highest number (53%) of BI-negative leprosy patients amongst all the gene targets used in this study. Amongst all the gene targets used in this study, PCR positivity using RLEP gene target was the highest in all the clinical and environmental samples. Further, the RLEP gene target was able to detect 53% of blood samples as positive in BI-negative leprosy cases indicating its future standardization and use for diagnostic purposes. Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  11. Archival Bone Marrow Samples: Suitable for Multiple Biomarker Analysis?

    DEFF Research Database (Denmark)

    Lund, Bendik; Najmi, A. Laeya; Wesolowska, Agata

    2015-01-01

    biopsies from 18 Danish and Norwegian childhood acute lymphoblastic leukemia patients were included and compared with corresponding blood samples. Samples were grouped according to the age of sample and whether WGA was performed or not. We found that measurements of DNA concentration after DNA extraction...

  12. Comparative analysis of five DNA isolation protocols and three drying methods for leaves samples of Nectandra megapotamica (Spreng. Mez

    Directory of Open Access Journals (Sweden)

    Leonardo Severo da Costa

    2016-06-01

    Full Text Available The aim of the study was to establish a DNA isolation protocol Nectandra megapotamica (Spreng. Mez., able to obtain samples of high yield and quality for use in genomic analysis. A commercial kit and four classical methods of DNA extraction were tested, including three cetyltrimethylammonium bromide (CTAB-based and one sodium dodecyl sulfate (SDS-based methods. Three drying methods for leaves samples were also evaluated including drying at room temperature (RT, in an oven at 40ºC (S40, and in a microwave oven (FMO. The DNA solutions obtained from different types of leaves samples using the five protocols were assessed in terms of cost, execution time, and quality and yield of extracted DNA. The commercial kit did not extract DNA with sufficient quantity or quality for successful PCR reactions. Among the classic methods, only the protocols of Dellaporta and of Khanuja yielded DNA extractions for all three types of foliar samples that resulted in successful PCR reactions and subsequent enzyme restriction assays. Based on the evaluated variables, the most appropriate DNA extraction method for Nectandra megapotamica (Spreng. Mez. was that of Dellaporta, regardless of the method used to dry the samples. The selected method has a relatively low cost and total execution time. Moreover, the quality and quantity of DNA extracted using this method was sufficient for DNA sequence amplification using PCR reactions and to get restriction fragments.

  13. Concordance of Hypermethylated DNA and the Tumor Markers CA 15-3, CEA, and TPA in Serum during Monitoring of Patients with Advanced Breast Cancer

    Directory of Open Access Journals (Sweden)

    Søren Kristiansen

    2015-01-01

    Full Text Available The serological protein tumor markers CA 15-3, CEA, and TPA are frequently used to monitor tumor burden among metastatic breast cancer patients. Breast cancer is associated with global DNA hypomethylation and hypermethylation of some promoter regions. No monitoring study has yet investigated the interrelationship between protein tumor markers, the global DNA hypomethylation, and hypermethylated genes in serum from patients with advanced disease. Twenty-nine patients with histologically proven advanced breast cancer received first-line chemotherapy with epirubicin. Samples were collected prior to each treatment and prospectively analyzed for CA 15-3, CEA, and TPA. The same samples were retrospectively analyzed for the concentration of hypermethylated RASSF1A and for global DNA hypomethylation using LINE-1. Among patients with elevated concentrations of the protein markers, concordance could be observed between serial changes of the hypermethylated RASSF1A gene and the protein markers. Among patients with lower concentrations, RASSF1A could only be detected periodically. There was discordance between changes of the hypomethylated LINE-1 as compared to the protein markers. Circulating hypermethylated RASSF1A and protein markers may have similar kinetics during monitoring of tumor burden. Further investigations are needed to determine whether any of the hypermethylated DNA genes may provide predictive information during monitoring.

  14. Concordance of Hypermethylated DNA and the Tumor Markers CA 15-3, CEA, and TPA in Serum during Monitoring of Patients with Advanced Breast Cancer.

    Science.gov (United States)

    Kristiansen, Søren; Jørgensen, Lars Mønster; Hansen, Morten Høgh; Nielsen, Dorte; Sölétormos, György

    2015-01-01

    The serological protein tumor markers CA 15-3, CEA, and TPA are frequently used to monitor tumor burden among metastatic breast cancer patients. Breast cancer is associated with global DNA hypomethylation and hypermethylation of some promoter regions. No monitoring study has yet investigated the interrelationship between protein tumor markers, the global DNA hypomethylation, and hypermethylated genes in serum from patients with advanced disease. Twenty-nine patients with histologically proven advanced breast cancer received first-line chemotherapy with epirubicin. Samples were collected prior to each treatment and prospectively analyzed for CA 15-3, CEA, and TPA. The same samples were retrospectively analyzed for the concentration of hypermethylated RASSF1A and for global DNA hypomethylation using LINE-1. Among patients with elevated concentrations of the protein markers, concordance could be observed between serial changes of the hypermethylated RASSF1A gene and the protein markers. Among patients with lower concentrations, RASSF1A could only be detected periodically. There was discordance between changes of the hypomethylated LINE-1 as compared to the protein markers. Circulating hypermethylated RASSF1A and protein markers may have similar kinetics during monitoring of tumor burden. Further investigations are needed to determine whether any of the hypermethylated DNA genes may provide predictive information during monitoring.

  15. Failure to detect circulating DNA-anti-DNA complexes by four radioimmunological methods in patients with systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Izui, S; Lambert, P H; Miescher, P A [Hopital Cantonal Geneve (Switzerland)

    1977-12-01

    The presence of The DNA-anti-DNA complexes in sera from patients with systemic lupus erythematosus (SLE) was investigated by two new radioimmunoassays (RIA) developed for this purpose and by measuring the CLq and DNA binding activity of serum before and after treatment with DNAse. Two direct RIA developed in this study were based on the reactivity of (/sup 3/H)actinomycin D ((/sup 3/H)ACT-D) or solid-phase methylated bovine serum albumin (mBSA) with DNA-anti-DNA complexes. DNA-anti-DNA complexes prepared in vitro could be efficiently detected at various antigen-antibody ratios by these two RIA. Increased levels of circulating immune complexes as indicated by the CLq binding test were found in 52% of the SLE sera. However, the frequency of specific DNA-anti-DNA complexes detected in the SLE sera was very low. Only 6% of the sera exhibited an increased value deviating by more than three s.d. from the normal mean when tested with the (/sup 3/H)ACT-D binding RIA or the solid-phase mBSA RIA. On the other hand, there was no significant difference in the serum CLq or DNA binding activity after treatment with DNAse. These results suggest that DNA-anti-DNA complexes do not occur frequently in circulating blood and represent only a very small portion of the immune complexes detected in serum from patients with SLE.

  16. Trace DNA Sampling Success from Evidence Items Commonly Encountered in Forensic Casework.

    Science.gov (United States)

    Dziak, Renata; Peneder, Amy; Buetter, Alicia; Hageman, Cecilia

    2018-05-01

    Trace DNA analysis is a significant part of a forensic laboratory's workload. Knowing optimal sampling strategies and item success rates for particular item types can assist in evidence selection and examination processes and shorten turnaround times. In this study, forensic short tandem repeat (STR) casework results were reviewed to determine how often STR profiles suitable for comparison were obtained from "handler" and "wearer" areas of 764 items commonly submitted for examination. One hundred and fifty-five (155) items obtained from volunteers were also sampled. Items were analyzed for best sampling location and strategy. For casework items, headwear and gloves provided the highest success rates. Experimentally, eyeglasses and earphones, T-shirts, fabric gloves and watches provided the highest success rates. Eyeglasses and latex gloves provided optimal results if the entire surfaces were swabbed. In general, at least 10%, and up to 88% of all trace DNA analyses resulted in suitable STR profiles for comparison. © 2017 American Academy of Forensic Sciences.

  17. Quantitative Field Testing Rotylenchulus reniformis DNA from Metagenomic Samples Isolated Directly from Soil

    Science.gov (United States)

    Showmaker, Kurt; Lawrence, Gary W.; Lu, Shien; Balbalian, Clarissa; Klink, Vincent P.

    2011-01-01

    A quantitative PCR procedure targeting the β-tubulin gene determined the number of Rotylenchulus reniformis Linford & Oliveira 1940 in metagenomic DNA samples isolated from soil. Of note, this outcome was in the presence of other soil-dwelling plant parasitic nematodes including its sister genus Helicotylenchus Steiner, 1945. The methodology provides a framework for molecular diagnostics of nematodes from metagenomic DNA isolated directly from soil. PMID:22194958

  18. Validation and clinical application of a nested PCR for paracoccidioidomycosis diagnosis in clinical samples from Colombian patients.

    Science.gov (United States)

    Gaviria, Marcela; Rivera, Vanessa; Muñoz-Cadavid, Cesar; Cano, Luz Elena; Naranjo, Tonny Williams

    2015-01-01

    Paracoccidioidomycosis is a systemic and endemic mycosis, restricted to tropical and subtropical areas of Latin America. The infection is caused by the thermal dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. The diagnosis of paracoccidioidomycosis is usually performed by microscopic examination, culture and immunodiagnostic tests to respiratory specimens, body fluids and/or biopsies; however these methods require laboratory personnel with experience and several days to produce a result. In the present study, we have validated and evaluated a nested PCR assay targeting the gene encoding the Paracoccidioides gp43 membrane protein in 191 clinical samples: 115 samples from patients with proven infections other than paracoccidioidomycosis, 51 samples as negative controls, and 25 samples from patients diagnosed with paracoccidioidomycosis. Additionally, the specificity of the nested PCR assay was also evaluated using purified DNA isolated from cultures of different microorganisms (n=35) previously identified by culture and/or sequencing. The results showed that in our hands, this nested PCR assay for gp43 protein showed specificity and sensitivity rates of 100%. The optimized nested PCR conditions in our laboratory allowed detection down to 1fg of P. brasiliensis DNA. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

  19. Sensitive detection of Treponema pallidum DNA from the whole blood of patients with syphilis by the nested PCR assay.

    Science.gov (United States)

    Wang, Cuini; Cheng, Yuanyuan; Liu, Biao; Wang, Yuanyuan; Gong, Weiming; Qian, Yihong; Guan, Zhifang; Lu, Haikong; Gu, Xin; Shi, Mei; Zhou, Pingyu

    2018-05-09

    The aim of this work was to investigate the application of the nested PCR assay for the detection of Treponema pallidum (TP) DNA from the blood of patients with different stages of syphilis. In this study, a nested PCR method targeting the Tpp47 and polA genes (Tpp47-Tp-PCR and polA-Tp-PCR) was developed to detect TP-DNA in whole blood samples collected from 262 patients with different stages of syphilis (84 primary syphilis, 97 secondary syphilis, and 81 latent syphilis patients). The PCR assay detected T. pallidum DNA in 53.6% and 62.9% of the patients with primary and secondary syphilis, respectively, which was much higher than the detection levels in patients with latent syphilis (7.4%) (both p PCR in the early phase of the latent infection. Thus, blood RPR titers were correlated with the blood T. pallidum burden, but the correlations varied with primary and secondary syphilis. The results indicate that nested PCR is a sensitive method for detecting blood TP-DNA and is especially useful for detecting early syphilis including primary syphilis and secondary syphilis. The findings also suggest that the PCR assay may be used to complement other methods to enhance the diagnosis of syphilis.

  20. Genetic mutation analysis of HBV covalently closed circular DNA in peripheral blood mononuclear cells from chronic hepatitis B patients with nucleos(tide analog-resistant mutations in serum virions

    Directory of Open Access Journals (Sweden)

    Zhong-bin LI

    2012-06-01

    Full Text Available Objective  To analyze the characteristics of genetic mutations in reverse-transcriptase (RT domain of HBV covalently closed circular DNA (cccDNA in peripheral blood mononuclear cells (PBMCs obtained from chronic hepatitis B (CHB patients with drug-resistant mutations in serum virions during nucleoside/nucleotide analog (NA therapy. Methods  A total of 30 CHB patients admitted to 302 Hospital of PLA from July 2010 to August 2011 were included in this study. All the patients were confirmed to harbor the drug-resistant mutations in serum virions during an NA therapy longer than 6 months. Total DNA was extracted from PBMCs isolated from 30 whole blood samples at the same time point as that of serum analysis. Plasmid-safe ATP-dependent DNase (PSAD digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR were used to amplify the RT region of HBV cccDNA. NA-resistant-associated mutations were analyzed at nine sites. Results  HBV cccDNA was efficiently amplified in 16 out of 30 (53.3% PBMC samples, and the detection rate was not correlated with HBeAg-positive rate, serum ALT level or HBV DNA load. Five of 16 (31.3% patients were sustained to have genotype B HBV infection, and 11 of 16 (68.8% were of genotype C HBV infection, and the result was consistent with the genotyping results using serum HBV. Different from drug-resistant mutations detected in the serum virions, the viruses detected in HBV cccDNA of 16 PBMC samples were all wild-type viruses without NA-resistant-associated mutations in RT region. Conclusions  During NA antiviral treatment, if drug-resistant mutations occur in serum HBV DNA of CHB patients, the dominant species of HBV cccDNA in PBMCs from the same patient is still the original wild-type strains. It is speculated that PBMCs might be the potential "repository" of HBV wild-type strain in vivo.

  1. Advantages and Limitations of Ribosomal RNA PCR and DNA Sequencing for Identification of Bacteria in Cardiac Valves of Danish Patients

    DEFF Research Database (Denmark)

    Kemp, Michael; Bangsborg, Jette; Kjerulf, Anne

    2013-01-01

    of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements. In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part...... of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between...... bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group. Ribosomal PCR with subsequent...

  2. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    Directory of Open Access Journals (Sweden)

    Ram, S.

    2012-01-01

    Full Text Available Aim: Isolation, dark field detection and microscopic agglutination test (MAT are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207 blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21% out of 100 culture positive blood samples, three (2.8% out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001. A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001 signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

  3. Intra-tumoral Heterogeneity of KRAS and BRAF Mutation Status in Patients with Advanced Colorectal Cancer (aCRC and Cost-Effectiveness of Multiple Sample Testing

    Directory of Open Access Journals (Sweden)

    Susan D. Richman

    2011-01-01

    Full Text Available KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor. We also investigated the utility and efficiency of genotyping a ‘DNA cocktail’ prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1% showed intratumoral heterogeneity; 5 (7.2% at KRAS codons 12, 13 and 2 (2.9% at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in ‘DNA cocktail’ samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a ‘DNA cocktail’ from two or more tumor blocks, improves mutation detection at minimal extra cost.

  4. DNA damages induced in human lymphocytes by UV or X-rays and repair capacities of healthy donors and skin cancer patients

    International Nuclear Information System (INIS)

    Cebulska-Wasilewska, A.; Dyga, W.; Budzanowska, E.

    1999-01-01

    The aim of this study was to compare variation in the individual susceptibility of various donors to the induction of the DNA damage by genotoxic agents and their cellular capabilities to repair induced damage. DNA damages induced by UV or X-rays in lymphocytes and cellular repair capability of healthy donors and persons bearing various categories of skin cancer cells were investigated. Fresh blood was collected by venipuncture from 35 individuals (including nine prior to skin cancer treatment). All cancer patients were nonsmoking males, however 42.3 % of them were former smokers. All healthy donors were also males, an average age was 38.6 y and among them 68% were recent or former smokers. Immediately after collecting samples, lymphocytes were isolated and stored at -70 o C for further studies in vitro. Previously cryopreserved lymphocytes were defrosted and viability of the cells was investigated. The single cell gel electrophoresis assay (SCGE), known as a Comet assay, was performed in defrozen lymphocytes to evaluate individual DNA damage levels presented in lymphocytes at the time of sample's collection. To compare individual susceptibility to the induction of DNA damage by UV and ionizing radiation, lymphocytes were exposed to dose of 6 J/m 2 of UV or 2 Gy of X-rays and DNA damages were detected again with an application of the Comet assay. Additionally, to study variation in the individuals cellular capability to repair damages induced, prior to the DNA damage analysis an incubation of cells exposed was also done in presence or absence of phytohemagglutinin (cell divisions processes starting agent). Results showed in untreated lymphocytes of skin cancer patients significantly higher than in the reference group levels of the DNA damages. Significantly different responses to UV and significantly lower capabilities to repair UV induced damage in skin cancer patients were observed. On the average, no differences between reference group and skin cancer patients

  5. Environmental DNA method for estimating salamander distribution in headwater streams, and a comparison of water sampling methods.

    Science.gov (United States)

    Katano, Izumi; Harada, Ken; Doi, Hideyuki; Souma, Rio; Minamoto, Toshifumi

    2017-01-01

    Environmental DNA (eDNA) has recently been used for detecting the distribution of macroorganisms in various aquatic habitats. In this study, we applied an eDNA method to estimate the distribution of the Japanese clawed salamander, Onychodactylus japonicus, in headwater streams. Additionally, we compared the detection of eDNA and hand-capturing methods used for determining the distribution of O. japonicus. For eDNA detection, we designed a qPCR primer/probe set for O. japonicus using the 12S rRNA region. We detected the eDNA of O. japonicus at all sites (with the exception of one), where we also observed them by hand-capturing. Additionally, we detected eDNA at two sites where we were unable to observe individuals using the hand-capturing method. Moreover, we found that eDNA concentrations and detection rates of the two water sampling areas (stream surface and under stones) were not significantly different, although the eDNA concentration in the water under stones was more varied than that on the surface. We, therefore, conclude that eDNA methods could be used to determine the distribution of macroorganisms inhabiting headwater systems by using samples collected from the surface of the water.

  6. Oxidative Stress, DNA Damage and DNA Repair in Female Patients with Diabetes Mellitus Type 2.

    Directory of Open Access Journals (Sweden)

    Annemarie Grindel

    Full Text Available Diabetes mellitus type 2 (T2DM is associated with oxidative stress which in turn can lead to DNA damage. The aim of the present study was to analyze oxidative stress, DNA damage and DNA repair in regard to hyperglycemic state and diabetes duration.Female T2DM patients (n = 146 were enrolled in the MIKRODIAB study and allocated in two groups regarding their glycated hemoglobin (HbA1c level (HbA1c≤7.5%, n = 74; HbA1c>7.5%, n = 72. In addition, tertiles according to diabetes duration (DD were created (DDI = 6.94±3.1 y, n = 49; DDII = 13.35±1.1 y, n = 48; DDIII = 22.90±7.3 y, n = 49. Oxidative stress parameters, including ferric reducing ability potential, malondialdehyde, oxidized and reduced glutathione, reduced thiols, oxidized LDL and F2-Isoprostane as well as the activity of antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase were measured. Damage to DNA was analyzed in peripheral blood mononuclear cells and whole blood with single cell gel electrophoresis. DNA base excision repair capacity was tested with the modified comet repair assay. Additionally, mRNA expressions of nine genes related to base excision repair were analyzed in a subset of 46 matched individuals.No significant differences in oxidative stress parameters, antioxidant enzyme activities, damage to DNA and base excision repair capacity, neither between a HbA1c cut off />7.5%, nor between diabetes duration was found. A significant up-regulation in mRNA expression was found for APEX1, LIG3 and XRCC1 in patients with >7.5% HbA1c. Additionally, we observed higher total cholesterol, LDL-cholesterol, LDL/HDL-cholesterol, triglycerides, Framingham risk score, systolic blood pressure, BMI and lower HDL-cholesterol in the hyperglycemic group.BMI, blood pressure and blood lipid status were worse in hyperglycemic individuals. However, no major disparities regarding oxidative stress, damage to DNA and DNA repair were present which might be due to good medical

  7. Patient-Specific Circulating Tumor DNA Detection during Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

    Science.gov (United States)

    Riva, Francesca; Bidard, Francois-Clement; Houy, Alexandre; Saliou, Adrien; Madic, Jordan; Rampanou, Aurore; Hego, Caroline; Milder, Maud; Cottu, Paul; Sablin, Marie-Paule; Vincent-Salomon, Anne; Lantz, Olivier; Stern, Marc-Henri; Proudhon, Charlotte; Pierga, Jean-Yves

    2017-03-01

    In nonmetastatic triple-negative breast cancer (TNBC) patients, we investigated whether circulating tumor DNA (ctDNA) detection can reflect the tumor response to neoadjuvant chemotherapy (NCT) and detect minimal residual disease after surgery. Ten milliliters of plasma were collected at 4 time points: before NCT; after 1 cycle; before surgery; after surgery. Customized droplet digital PCR (ddPCR) assays were used to track tumor protein p53 ( TP53 ) mutations previously characterized in tumor tissue by massively parallel sequencing (MPS). Forty-six patients with nonmetastatic TNBC were enrolled. TP53 mutations were identified in 40 of them. Customized ddPCR probes were validated for 38 patients, with excellent correlation with MPS ( r = 0.99), specificity (≥2 droplets/assay), and sensitivity (at least 0.1%). At baseline, ctDNA was detected in 27/36 patients (75%). Its detection was associated with mitotic index ( P = 0.003), tumor grade ( P = 0.003), and stage ( P = 0.03). During treatment, we observed a drop of ctDNA levels in all patients but 1. No patient had detectable ctDNA after surgery. The patient with rising ctDNA levels experienced tumor progression during NCT. Pathological complete response (16/38 patients) was not correlated with ctDNA detection at any time point. ctDNA positivity after 1 cycle of NCT was correlated with shorter disease-free ( P < 0.001) and overall ( P = 0.006) survival. Customized ctDNA detection by ddPCR achieved a 75% detection rate at baseline. During NCT, ctDNA levels decreased quickly and minimal residual disease was not detected after surgery. However, a slow decrease of ctDNA level during NCT was strongly associated with shorter survival. © 2016 American Association for Clinical Chemistry.

  8. Application of the CometChip platform to assess DNA damage in field-collected blood samples from turtles.

    Science.gov (United States)

    Sykora, Peter; Chiari, Ylenia; Heaton, Andrew; Moreno, Nickolas; Glaberman, Scott; Sobol, Robert W

    2018-05-01

    DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  9. Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ® swabs.

    Science.gov (United States)

    Ambers, Angie; Wiley, Rachel; Novroski, Nicole; Budowle, Bruce

    2018-01-01

    Previous studies have shown that nylon flocked swabs outperform traditional fiber swabs in DNA recovery due to their innovative design and lack of internal absorbent core to entrap cellular materials. The microFLOQ ® Direct swab, a miniaturized version of the 4N6 FLOQSwab ® , has a small swab head that is treated with a lysing agent which allows for direct amplification and DNA profiling from sample collection to final result in less than two hours. Additionally, the microFLOQ ® system subsamples only a minute portion of a stain and preserves the vast majority of the sample for subsequent testing or re-analysis, if desired. The efficacy of direct amplification of DNA from dilute bloodstains, saliva stains, and touch samples was evaluated using microFLOQ ® Direct swabs and the GlobalFiler™ Express system. Comparisons were made to traditional methods to assess the robustness of this alternate workflow. Controlled studies with 1:19 and 1:99 dilutions of bloodstains and saliva stains consistently yielded higher STR peak heights than standard methods with 1ng input DNA from the same samples. Touch samples from common items yielded single source and mixed profiles that were consistent with primary users of the objects. With this novel methodology/workflow, no sample loss occurs and therefore more template DNA is available during amplification. This approach may have important implications for analysis of low quantity and/or degraded samples that plague forensic casework. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Accuracy of qPCR for quantifying Leishmania kDNA in different skin layers of patients with American tegumentary leishmaniasis.

    Science.gov (United States)

    Sevilha-Santos, L; Dos Santos Júnior, A C M; Medeiros-Silva, V; Bergmann, J O; da Silva, E F; Segato, L F; Arabi, A Y M; de Paula, N A; Sampaio, R N R; Lima, B D; Gomes, C M

    2018-05-03

    Superficial swab sampling of American tegumentary leishmaniasis (ATL) lesions shows higher amounts of Leishmania than those from biopsy. Subcutaneous involvement is also important in ATL, but parasite quantification according to lesion depth has not been evaluated. We aim to present the best depth at which sampling should be performed for molecular exams of ATL. Patients with a clinical presentation compatible with ATL were allocated to ATL and control groups. Qualitative and quantitative qPCR assays were performed using SYBR Green and primers amplifying the kDNA minicircle of Leishmania spp. in different skin layers, including the epidermis, the superior dermis, the inferior dermis, and the hypodermis. Fifty-nine patients were included in this study, including 40 who had been diagnosed with ATL and 19 controls. The number of parasites was greater in samples of the epidermis and superior dermis (159.1 × 10 6 , range 4.0-781.7, and 75.4 × 10 6 , range 8.0-244.5, mean Leishmania parasite equivalents per μg of tissue DNA, respectively) than those in samples of the inferior dermis and hypodermis (54.6, range 8.0-256.6, and 16.8 × 10 6 , range 8.0-24.1, mean Leishmania parasite equivalents per μg of tissue DNA, respectively). The best diagnostic accuracy was achieved in the superior dermis (77.9%) and was significantly greater than that in the hypodermis (63.3%; p 0.039). We conclude that superficial sampling can retrieve a greater quantity of parasites. Future studies of the role of transepidermal elimination as a mechanism of host defence in ATL must be performed as there is a considerable quantity of Leishmania kDNA in the epidermis. Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  11. DNA-methylation profiling of fetal tissues reveals marked epigenetic differences between chorionic and amniotic samples.

    Directory of Open Access Journals (Sweden)

    Christel Eckmann-Scholz

    Full Text Available Epigenetic mechanisms including DNA methylation are supposed to play a key role in fetal development. Here we have investigated fetal DNA-methylation levels of 27,578 CpG loci in 47 chorionic villi (CVS and 16 amniotic cell (AC samples. Methylation levels differed significantly between karyotypically normal AC and CVS for 2,014 genes. AC showed more extreme DNA-methylation levels of these genes than CVS and the differentially methylated genes are significantly enriched for processes characteristic for the different cell types sampled. Furthermore, we identified 404 genes differentially methylated in CVS with trisomy 21. These genes were significantly enriched for high CG dinucleotid (CpG content and developmental processes associated with Down syndrome. Our study points to major tissue-specific differences of fetal DNA-methylation and gives rise to the hypothesis that part of the Down syndrome phenotype is epigenetically programmed in the first trimester of pregnancy.

  12. Comparison of DNA preservation methods for environmental bacterial community samples.

    Science.gov (United States)

    Gray, Michael A; Pratte, Zoe A; Kellogg, Christina A

    2013-02-01

    Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard(™), RNAlater(®), DMSO-EDTA-salt (DESS), FTA(®) cards, and FTA Elute(®) cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA(®) cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard(™), RNAlater(®), and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  13. Association of mitochondrial DNA in peripheral blood with depression, anxiety and stress- and adjustment disorders in primary health care patients.

    Science.gov (United States)

    Wang, Xiao; Sundquist, Kristina; Rastkhani, Hamideh; Palmér, Karolina; Memon, Ashfaque A; Sundquist, Jan

    2017-08-01

    Mitochondrial dysfunction may result in a variety of diseases. The objectives here were to examine possible differences in mtDNA copy number between healthy controls and patients with depression, anxiety or stress- and adjustment disorders; the association between mtDNA copy number and disease severity at baseline; and the association between mtDNA copy number and response after an 8-week treatment (mindfulness, cognitive based therapy). A total of 179 patients in primary health care (age 20-64 years) with depression, anxiety and stress- and adjustment disorders, and 320 healthy controls (aged 19-70 years) were included in the study. Relative mtDNA copy number was measured using quantitative real-time PCR on peripheral blood samples. We found that the mean mtDNA copy number was significantly higher in patients compared to controls (84.9 vs 75.9, pAnxiety and Depression Scale (HADS-D) and PHQ-9 scores (ß=1.00, p=0.03 and ß=0.65, p=0.04, respectively), after controlling for baseline scores, age, sex, BMI, smoking status, alcohol drinking and medication. Our findings show that mtDNA copy number is associated with symptoms of depression, anxiety and stress- and adjustment disorders and treatment response in these disorders. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  14. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.

    Science.gov (United States)

    Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew

    2017-11-06

    Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.

  15. A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

    Science.gov (United States)

    Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

    2017-01-01

    A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

  16. Insights into the processes behind the contamination of degraded human teeth and bone samples with exogenous sources of DNA

    DEFF Research Database (Denmark)

    Gilbert, M. T. P.; Hansen, Anders J.; Willerslev, E.

    2006-01-01

    A principal problem facing human DNA studies that use old and degraded remains is contamination from other sources of human DNA. In this study we have attempted to contaminate deliberately bones and teeth sampled from a medieval collection excavated in Trondheim, Norway, in order to investigate......, prior to assaying for the residual presence of the handler's DNA. Surprisingly, although our results suggest that a large proportion of the teeth were contaminated with multiple sources of human DNA prior to our investigation, we were unable to contaminate the samples with further human DNA. One...

  17. Decreased expression level of BER genes in Alzheimer's disease patients is not derivative of their DNA methylation status.

    Science.gov (United States)

    Sliwinska, Agnieszka; Sitarek, Przemysław; Toma, Monika; Czarny, Piotr; Synowiec, Ewelina; Krupa, Renata; Wigner, Paulina; Bialek, Katarzyna; Kwiatkowski, Dominik; Korycinska, Anna; Majsterek, Ireneusz; Szemraj, Janusz; Galecki, Piotr; Sliwinski, Tomasz

    2017-10-03

    Neurodegeneration in Alzheimer's disease can be caused by accumulation of oxidative DNA damage resulting from altered expression of genes involved in the base excision repair system (BER). Promoter methylation can affect the profile of BER genes expression. Decreased expression of BER genes was observed in the brains of AD patients. The aim of our study was to compare the expression and methylation profiles of six genes coding for proteins involved in BER, namely: hOGG1, APE1, MUTYH, NEIL1, PARP1 and XRCC1, in the peripheral blood cells of AD patients and healthy volunteers. The study consisted of 100 persons diagnosed with Alzheimer's disease according to DSM-IV criteria, and 110 healthy volunteers. DNA and total RNA were isolated from venous blood cells. Promoter methylation profiles were obtained by High Resolution Melting (HRM) analysis of bisulfide converted DNA samples. Real-time PCR with TaqMan probes was employed for gene expression analysis. APE1, hOGG1, MUTYH, PARP1 and NEIL1 were significantly (pgenes. The methylation status of promoters is not associated with downregulation of BER genes. Our results show that downregulation of BER genes detected in peripheral blood samples could reflect the changes occurring in the brain of patients with AD, and may be a useful biomarker of this disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. SPIDIA-DNA: An External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses

    Czech Academy of Sciences Publication Activity Database

    Malentacchi, F.; Pazzagli, M.; Simi, L.; Orlando, C.; Wyrich, R.; Hartmann, C.C.; Verderio, P.; Pizzamiglio, S.; Ciniselli, C.M.; Tichopád, Aleš; Kubista, Mikael; Gelmini, S.

    -, č. 424 (2013), s. 274-286 ISSN 0009-8981 Institutional research plan: CEZ:AV0Z50520701 Keywords : Pre-analytical phase * DNA quality * Blood samples Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.764, year: 2013

  19. Rapid colorimetric assay for detection of Listeria monocytogenes in food samples using LAMP formation of DNA concatemers and gold nanoparticle-DNA probe complex

    Science.gov (United States)

    Wachiralurpan, Sirirat; Sriyapai, Thayat; Areekit, Supatra; Sriyapai, Pichapak; Augkarawaritsawong, Suphitcha; Santiwatanakul, Somchai; Chansiri, Kosum

    2018-04-01

    ABSTRACT Listeria monocytogenes is a major foodborne pathogen of global health concern. Herein, the rapid diagnosis of L. monocytogenes has been achieved using loop-mediated isothermal amplification (LAMP) based on the phosphatidylcholine-phospholipase C gene (plcB). Colorimetric detection was then performed through the formation of DNA concatemers and a gold nanoparticle/DNA probe complex (GNP/DNA probe). The overall detection process was accomplished within approximately 1 h with no need for complicated equipment. The limits of detection for L. monocytogenes in the forms of purified genomic DNA and pure culture were 800 fg and 2.82 CFU mL-1, respectively. No cross reactions were observed from closely related bacteria species. The LAMP-GNP/DNA probe assay was applied to the detection of 200 raw chicken meat samples and compared to routine standard methods. The data revealed that the specificity, sensitivity and accuracy were 100%, 90.20% and 97.50%, respectively. The present assay was 100% in conformity with LAMP-agarose gel electrophoresis assay. Five samples that were negative by both assays appeared to have the pathogen at below the level of detection. The assay can be applied as a rapid direct screening method for L. monocytogenes.

  20. Analysis of RTEL1 and PCDHGB6 promoter methylation in circulating-free DNA of lung cancer patients using liquid biopsy: A pilot study.

    Science.gov (United States)

    Powrózek, Tomasz; Krawczyk, Paweł; Kuźnar-Kamińska, Barbara; Batura-Gabryel, Halina; Milanowski, Janusz

    2016-08-01

    Analysis of epigenetic alterations such as methylation of circulating-free DNA (cf-DNA) expression significantly broadened perspectives of lung cancer (LC) screening. Moreover, methylation of tumor suppressor genes may be analyzed with non-invasive manner in patients' blood samples (liquid biopsy), what underline necessity of detailed investigation of tumor cf-DNA. The purpose of current study was to assess methylation of RTEL1 and PCDHGB6 promoter regions in cf-DNA of 70 LC patients and 80 healthy individuals using qMSP-PCR technique. Methylation status of both genes has not been investigated in cf-DNA of LC patients before. PCDHGB6 promoter methylation was found in 41.4% of LC patients and in 1.3% of healthy individuals, whereas promoter of RTEL1 was found methylated in 51.4% of LC patients and in 8.8% of healthy individuals. Combined analysis of two markers improved test sensitivity up to 62.9% and specificity up to 90% with area under the curve (AUC) in receiver operating curve (ROC) of 0.755. The evaluation of RTEL1 and PCDHGB6 promoter methylation may be an useful tool for non-invasive diagnosis of LC in liquid biopsy.

  1. Coding of DNA samples and data in the pharmaceutical industry: current practices and future directions--perspective of the I-PWG.

    Science.gov (United States)

    Franc, M A; Cohen, N; Warner, A W; Shaw, P M; Groenen, P; Snapir, A

    2011-04-01

    DNA samples collected in clinical trials and stored for future research are valuable to pharmaceutical drug development. Given the perceived higher risk associated with genetic research, industry has implemented complex coding methods for DNA. Following years of experience with these methods and with addressing questions from institutional review boards (IRBs), ethics committees (ECs) and health authorities, the industry has started reexamining the extent of the added value offered by these methods. With the goal of harmonization, the Industry Pharmacogenomics Working Group (I-PWG) conducted a survey to gain an understanding of company practices for DNA coding and to solicit opinions on their effectiveness at protecting privacy. The results of the survey and the limitations of the coding methods are described. The I-PWG recommends dialogue with key stakeholders regarding coding practices such that equal standards are applied to DNA and non-DNA samples. The I-PWG believes that industry standards for privacy protection should provide adequate safeguards for DNA and non-DNA samples/data and suggests a need for more universal standards for samples stored for future research.

  2. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  3. Detection of plant DNA in the bronchoalveolar lavage of patients with ventilator-associated pneumonia.

    Directory of Open Access Journals (Sweden)

    Sabri Bousbia

    Full Text Available BACKGROUND: Hospital-acquired infections such as nosocomial pneumonia are a serious cause of mortality for hospitalized patients, especially for those admitted to intensive care units (ICUs. Despite the number of the studies reported to date, the causative agents of pneumonia are not completely known. Herein, we found by molecular technique that vegetable and tobacco DNA may be detected in the bronchoalveolar lavage from patients with ventilator-associated pneumonia (VAP. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we studied bronchoalveolar lavage (BAL from patients admitted to ICUs with ventilator-associated pneumonia. BAL fluids were assessed with molecular tests, culture and blood culture. We successfully identified plant DNA in six patients out of 106 (6% with ventilator-associated pneumonia. Inhalation was confirmed in four cases and suspected in the other two cases. Inhalation was significantly frequent in patients with plant DNA (four out of six patients than those without plant DNA (three out of 100 patients (P<0.001. Nicotiana tabacum chloroplast DNA was identified in three patients who were smokers (cases 2, 3 and 6. Cucurbita pepo, Morus bombycis and Triticum aestivum DNA were identified in cases 1, 4 and 5 respectively. Twenty-three different bacterial species, two viruses and five fungal species were identified from among these six patients by using molecular and culture techniques. Several of the pathogenic microorganisms identified are reported to be food-borne or tobacco plant-associated pathogens. CONCLUSIONS/SIGNIFICANCE: Our study shows that plants DNA may be identified in the BAL fluid of pneumonia patients, especially when exploring aspiration pneumonia, but the significance of the presence of plant DNA and its role in the pathogenesis of pneumonia is unknown and remains to be investigated. However, the identification of these plants may be a potential marker of aspiration in patients with pneumonia.

  4. Distribution and Prevalence of Myxobolus cerebralis in Postfire Areas of Plumas National Forest: Utility of Environmental DNA Sampling.

    Science.gov (United States)

    Richey, Christine A; Kenelty, Kirsten V; Van Stone Hopkins, Kristina; Stevens, Brittany N; Martínez-López, Beatriz; Barnum, Samantha M; Hallett, Sascha L; Atkinson, Stephen D; Bartholomew, Jerri L; Soto, Esteban

    2018-04-30

    Myxobolus cerebralis is a myxozoan parasite and the etiological agent of whirling disease in salmonids. The parasite's life cycle involves waterborne spores and requires both a salmonid fish and the benthic freshwater oligochaete worm Tubifex tubifex (Oligochaeta: Tubificidae). Wildfires can lead to the erosion of fine sediments into stream channels and have been implicated as promoting environmental conditions that are suitable for the survival and success of T. tubifex, whose presence in turn can affect the prevalence of M. cerebralis. Analysis of environmental DNA (eDNA) has the potential to be a powerful tool for evaluating the presence of microorganisms, for which direct observation is impossible. We investigated the presence of M. cerebraliseDNA in river water and river sediment samples collected from areas affected by recent fire activity in Plumas National Forest, California. We compared eDNA loads in the environment to M. cerebralis infection in T. tubifex and sentinel-exposed Rainbow Trout Oncorhynchus mykiss and the presence of T. tubifex lineages in the same environment. For the latter, we developed a multiplex quantitative PCR assay for detection of T. tubifex lineages I, III, and V. Lineage IIIT. tubifex and M. cerebralis (eDNA as well as DNA extracted from fish and worm tissues) were detected only in samples obtained from areas affected by the Moonlight wildfire. The association between M. cerebralis infection in sentinel-exposed fish and eDNA detection in environmental samples only approached significance at a P-value of 0.056. However, given the difference in relative effort between the two sampling methods (host versus nonhost environment), our data suggest that eDNA sampling of water and substrate is a promising approach for surveillance of myxozoan fish parasites. © 2018 American Fisheries Society.

  5. Study of Preoperative Antiviral Treatment of Patients with HCC Negative for HBV-DNA.

    Science.gov (United States)

    Liu, Xiao-Fang; Zhang, Tong; Tang, Kun; Sui, Lu-Lu; Xu, Gang; Liu, Qiang

    2017-08-01

    To study preoperative HBV-DNA negative HBV-related hepatocellular carcinoma (HCC) which was reactivated after surgery and could influence liver function and HCC recurrence. Patients were divided into two groups according to preoperative antiviral therapy status. The control group comprised of 102 preoperative HBV-DNA-negative patients who had not undergone antiviral therapy before surgery. In the treatment group, all HBV-DNA-negative patients (n=63) received entecavir 3-5 days before surgery and for 12 months after surgery. Patients were followed-up regularly, during the preoperative period, and at 1, 3, 6, 12, 18, 24, 30 and 36 months postoperatively. The data for the two groups were analyzed including the level of HBV-DNA and HBV-DNA activation; liver function; 1-, 2- and 3-year survival rate; cumulative survival time; and tumor recurrence. Liver function in the treatment group was better than that of the control group12 months after surgery. Compared to the control group, total bilirubin in the treatment group was significantly better at 6 and 12 months after surgery (pHBV-DNA activation while there were 13 cases (12.75%) with HBV-DNA activation in the control group (pHBV-related HCC with negative HBV-DNA is beneficial to liver function, coagulation function, disease control, prevention of tumor recurrence, improvement of patient quality of life, reduces the death rate and prolongs survival duration. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  6. Genetic Ancestry using Mitochondrial DNA in patients with Triple-negative breast cancer (GAMiT study).

    Science.gov (United States)

    Rao, Roshni; Rivers, Aeisha; Rahimi, Asal; Wooldridge, Rachel; Rao, Madhu; Leitch, Marilyn; Euhus, David; Haley, Barbara B

    2017-01-01

    Triple-negative breast cancer (TNBC) lacks estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2)/neu receptors, and is aggressive and therapeutically challenging. Genetic ancestry testing is an emerging medical field. Mitochondrial DNA (mtDNA), which is distinct from nuclear DNA, is maternally inherited and allows for origin determination. Patients with TNBC tend to be younger and are more likely to be African American, making this an ideal disease for mtDNA exploration. To the authors' knowledge, the current study is the first to perform mtDNA for self-described African American, White, and Hispanic patients with TNBC to identify mtDNA patterns. Patients with TNBC who were at any stage of therapy/survivorship were included. Self-reported ethnicity was confirmed at the time of the prospective buccal swab. Haplogroup prediction was performed on sequencing of hypervariable region 1. Using sequence similarity scores and lineage databases, sequence patterns were determined. Data regarding presentation and treatment, tumor features, and outcomes was collected. A total of 92 patients were included: 31 self-described African American, 31 White, and 30 Hispanic individuals. Hispanic patients were found to have the largest tumor size (4.5 cm; P = .01) and youngest age (41 years; Pancestry and haplogroups A, U, H, or B to be the most common mtDNA patterns. Twelve discordances (13%) between mtDNA analysis and self-described ethnicity were identified among the 92 patients. The highest discordance (26%; 8 patients) was noted in self-described Hispanic patients: 3 had Nigerian ancestry, and 1 individual demonstrated haplogroup K mtDNA (Ashkenazi Jewish ancestry). Discordance between self-reported ethnicity and mtDNA analysis was identified in 13% of patients with TNBC. The identification of mtDNA patterns with a predisposition toward TNBC may allow for risk stratification. Cancer 2017;107-113. © 2016 American Cancer Society. © 2016 American Cancer

  7. Solution-based targeted genomic enrichment for precious DNA samples

    Directory of Open Access Journals (Sweden)

    Shearer Aiden

    2012-05-01

    Full Text Available Abstract Background Solution-based targeted genomic enrichment (TGE protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1 modifying or eliminating time consuming steps; 2 increasing yield to reduce input DNA and excessive PCR cycling; and 3 enhancing reproducible. Results We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. Conclusions This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

  8. An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

    DEFF Research Database (Denmark)

    Bag, Satyabrata; Saha, Bipasa; Mehta, Ojasvi

    2016-01-01

    and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing...... methodologies and the supremacy of our method was confirmed. Maximum recovery of genomic DNA in the absence of substantial amount of impurities made the method convenient for nucleic acid extraction. The nucleic acids obtained using this method are suitable for different downstream applications. This improved...

  9. Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems

    Directory of Open Access Journals (Sweden)

    Camila Ximenes

    2014-12-01

    Full Text Available The Global Program for the Elimination of Lymphatic Filariasis (GPELF aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2 and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2, which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

  10. Evaluating sampling strategy for DNA barcoding study of coastal and inland halo-tolerant Poaceae and Chenopodiaceae: A case study for increased sample size.

    Science.gov (United States)

    Yao, Peng-Cheng; Gao, Hai-Yan; Wei, Ya-Nan; Zhang, Jian-Hang; Chen, Xiao-Yong; Li, Hong-Qing

    2017-01-01

    Environmental conditions in coastal salt marsh habitats have led to the development of specialist genetic adaptations. We evaluated six DNA barcode loci of the 53 species of Poaceae and 15 species of Chenopodiaceae from China's coastal salt marsh area and inland area. Our results indicate that the optimum DNA barcode was ITS for coastal salt-tolerant Poaceae and matK for the Chenopodiaceae. Sampling strategies for ten common species of Poaceae and Chenopodiaceae were analyzed according to optimum barcode. We found that by increasing the number of samples collected from the coastal salt marsh area on the basis of inland samples, the number of haplotypes of Arundinella hirta, Digitaria ciliaris, Eleusine indica, Imperata cylindrica, Setaria viridis, and Chenopodium glaucum increased, with a principal coordinate plot clearly showing increased distribution points. The results of a Mann-Whitney test showed that for Digitaria ciliaris, Eleusine indica, Imperata cylindrica, and Setaria viridis, the distribution of intraspecific genetic distances was significantly different when samples from the coastal salt marsh area were included (P Imperata cylindrica and Chenopodium album, average intraspecific distance tended to reach stability. These results indicate that the sample size for DNA barcode of globally distributed species should be increased to 11-15.

  11. Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

    Science.gov (United States)

    Salotra, Poonam; Sreenivas, G.; Pogue, Gregory P.; Lee, Nancy; Nakhasi, Hira L.; Ramesh, V.; Negi, N. S.

    2001-01-01

    We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples. PMID:11230394

  12. Comparison of three mycobacterial DNA extraction methods from extrapulmonary samples for PCR assay

    Directory of Open Access Journals (Sweden)

    Khandaker Shadia

    2012-01-01

    Full Text Available Sensitivity of the molecular diagnostic tests of extrapulmonary tuberculosis largely depends upon the efficiency of DNA extraction methods. The objective of our study was to compare three methods of extracting DNA of Mycobacterium tuberculosis for testing by polymerase chain reaction. All three methods; heating, heating with sonication and addition of lysis buffer with heating and sonication were implicated on 20 extrapulmonary samples. PCR positivity was 2 (10%, 4 (20% and 7 (35% in the samples extracted by heating, heat+sonication and heat+sonication+lysis buffer method respectively. Of the extraction methods evaluated, maximum PCR positive results were achieved by combined heat, sonication and lysis buffer method which can be applied in routine clinical practice. Ibrahim Med. Coll. J. 2012; 6(1: 9-11

  13. Patient identification in blood sampling.

    Science.gov (United States)

    Davidson, Anne; Bolton-Maggs, Paula

    The majority of adverse reports relating to blood transfusions result from human error, including misidentification of patients and incorrect labelling of samples. This article outlines best practice in blood sampling for transfusion (but is recommended for all pathology samples) and the role of patient empowerment in improving safety.

  14. Serial circulating tumour DNA analysis during multimodality treatment of locally advanced rectal cancer: a prospective biomarker study.

    Science.gov (United States)

    Tie, Jeanne; Cohen, Joshua D; Wang, Yuxuan; Li, Lu; Christie, Michael; Simons, Koen; Elsaleh, Hany; Kosmider, Suzanne; Wong, Rachel; Yip, Desmond; Lee, Margaret; Tran, Ben; Rangiah, David; Burge, Matthew; Goldstein, David; Singh, Madhu; Skinner, Iain; Faragher, Ian; Croxford, Matthew; Bampton, Carolyn; Haydon, Andrew; Jones, Ian T; S Karapetis, Christos; Price, Timothy; Schaefer, Mary J; Ptak, Jeanne; Dobbyn, Lisa; Silliman, Natallie; Kinde, Isaac; Tomasetti, Cristian; Papadopoulos, Nickolas; Kinzler, Kenneth; Volgestein, Bert; Gibbs, Peter

    2018-02-02

    For patients with locally advanced rectal cancer (LARC), adjuvant chemotherapy selection following surgery remains a major clinical dilemma. Here, we investigated the ability of circulating tumour DNA (ctDNA) to improve risk stratification in patients with LARC. We enrolled patients with LARC (T3/T4 and/or N+) planned for neoadjuvant chemoradiotherapy. Plasma samples were collected pretreatment, postchemoradiotherapy and 4-10 weeks after surgery. Somatic mutations in individual patient's tumour were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. We then designed personalised assays to quantify ctDNA in plasma samples. Patients received adjuvant therapy at clinician discretion, blinded to the ctDNA results. We analysed 462 serial plasma samples from 159 patients. ctDNA was detectable in 77%, 8.3% and 12% of pretreatment, postchemoradiotherapy and postsurgery plasma samples. Significantly worse recurrence-free survival was seen if ctDNA was detectable after chemoradiotherapy (HR 6.6; Pguide patient selection for adjuvant chemotherapy. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  15. A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples.

    Science.gov (United States)

    Zhou, Liqing; Pollard, Andrew J

    2012-07-27

    Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease. Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target. Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold. Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of

  16. C-14 bomb peak dating of human DNA samples at the microgram level

    International Nuclear Information System (INIS)

    Liebl, J. C.

    2011-01-01

    Radiocarbon (14C, t 1/2 = 5700 ± 30 years) is probably the radionuclide with the most versatile applications, spanning from archaeology to geoscience and medicine. Many of these applications are finally limited by the minimum amount of carbon in which the isotopic ratio 14C/12C can be measured. The required carbon sample size has dramatically decreased with the development of Accelerator Mass Spectrometry (AMS), typically from gram amounts for the classical beta counting method to about 1 milligram for AMS. The current work presents a further reduction into the few-μg carbon range. This means a decrease by a factor of one million compared to classical beta counting and is essential for the field of retrospective birth dating of human cells by means of radiocarbon from above-ground nuclear weapons testing between 1955 and 1963. The determination of 14C levels in genomic DNA can be used to retrospectively establish the birth date of cells in the human body. The main motive of the current work was to reduce the amount of carbon required for reliable 14C measurements to such an extent that investigations of neurons of particularly interesting small sections of the human brain (e.g. the olfactory bulb, bulbus olfactorius) were possible. In-depth investigations and development of 14C AMS sample preparation and measurement methods for μg-size DNA samples were carried out in close collaboration with the Department of Cell and Molecular Biology of the Karolinska Institute in Stockholm. As the most significant result, 14C measurements of 4.6 μg carbon DNA samples were performed with an overall precision of 2.3%. This allowed to study neurogenesis in the human olfactory bulb, which turned out to take place primarily at birth. Assuming throughout life a constant annual renewal rate of neurons in the human olfactory bulb, an upper limit of 0.34% for the renewal rate (95% confidence) was determined. At the Vienna Environmental Research Accelerator (VERA) the μg carbon

  17. Urinary Cell-Free DNA Quantification as Non-Invasive Biomarker in Patients with Bladder Cancer.

    Science.gov (United States)

    Brisuda, Antonin; Pazourkova, Eva; Soukup, Viktor; Horinek, Ales; Hrbáček, Jan; Capoun, Otakar; Svobodova, Iveta; Pospisilova, Sarka; Korabecna, Marie; Mares, Jaroslav; Hanuš, Tomáš; Babjuk, Marek

    2016-01-01

    Concentration of urinary cell-free DNA (ucfDNA) belongs to potential bladder cancer markers, but the reported results are inconsistent due to the use of various non-standardised methodologies. The aim of the study was to standardise the methodology for ucfDNA quantification as a potential non-invasive tumour biomarker. In total, 66 patients and 34 controls were enrolled into the study. Volumes of each urine portion (V) were recorded and ucfDNA concentrations (c) were measured using real-time PCR. Total amounts (TA) of ucfDNA were calculated and compared between patients and controls. Diagnostic accuracy of the TA of ucfDNA was determined. The calculation of TA of ucfDNA in the second urine portion was the most appropriate approach to ucfDNA quantification, as there was logarithmic dependence between the volume and the concentration of a urine portion (p = 0.0001). Using this methodology, we were able to discriminate between bladder cancer patients and subjects without bladder tumours (p = 0.0002) with area under the ROC curve of 0.725. Positive and negative predictive value of the test was 90 and 45%, respectively. Quantification of ucf DNA according to our modified method could provide a potential non-invasive biomarker for diagnosis of patients with bladder cancer. © 2015 S. Karger AG, Basel.

  18. Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

    Science.gov (United States)

    Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

    2018-06-01

    Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

  19. DNA Profiling of Convicted Offender Samples for the Combined DNA Index System

    Science.gov (United States)

    Millard, Julie T

    2011-01-01

    The cornerstone of forensic chemistry is that a perpetrator inevitably leaves trace evidence at a crime scene. One important type of evidence is DNA, which has been instrumental in both the implication and exoneration of thousands of suspects in a wide range of crimes. The Combined DNA Index System (CODIS), a network of DNA databases, provides…

  20. Simultaneous assessment of the macrobiome and microbiome in a bulk sample of tropical arthropods through DNA metasystematics.

    Science.gov (United States)

    Gibson, Joel; Shokralla, Shadi; Porter, Teresita M; King, Ian; van Konynenburg, Steven; Janzen, Daniel H; Hallwachs, Winnie; Hajibabaei, Mehrdad

    2014-06-03

    Conventional assessments of ecosystem sample composition are based on morphology-based or DNA barcode identification of individuals. Both approaches are costly and time-consuming, especially when applied to the large number of specimens and taxa commonly included in ecological investigations. Next-generation sequencing approaches can overcome the bottleneck of individual specimen isolation and identification by simultaneously sequencing specimens of all taxa in a bulk mixture. Here we apply multiple parallel amplification primers, multiple DNA barcode markers, 454-pyrosequencing, and Illumina MiSeq sequencing to the same sample to maximize recovery of the arthropod macrobiome and the bacterial and other microbial microbiome of a bulk arthropod sample. We validate this method with a complex sample containing 1,066 morphologically distinguishable arthropods from a tropical terrestrial ecosystem with high taxonomic diversity. Multiamplicon next-generation DNA barcoding was able to recover sequences corresponding to 91% of the distinguishable individuals in a bulk environmental sample, as well as many species present as undistinguishable tissue. 454-pyrosequencing was able to recover 10 more families of arthropods and 30 more species than did conventional Sanger sequencing of each individual specimen. The use of other loci (16S and 18S ribosomal DNA gene regions) also added the detection of species of microbes associated with these terrestrial arthropods. This method greatly decreases the time and money necessary to perform DNA-based comparisons of biodiversity among ecosystem samples. This methodology opens the door to much cheaper and increased capacity for ecological and evolutionary studies applicable to a wide range of socio-economic issues, as well as a basic understanding of how the world works.

  1. DNA-repair after UV-irradiation in skin fibroblasts from patients with actinic keratosis

    International Nuclear Information System (INIS)

    Sbano, E.; Andreassi, L.; Fimiani, M.; Valentino, A.; Baiocchi, R.

    1978-01-01

    Autoradiographic counting technique was utilized to measure the ultraviolet-induced unscheduled DNA synthesis of skin fibroblasts from 12 patients with chronic actinic keratosis and from 12 healthy donors of about the same age. In order to reveal a possible regional difference of DNA repair between the parts of the body ordinarily exposed and those parts unexposed to sunlight, two cell strains were used for each examined subject; one developed from the forehead skin and the other from the abdominal or axillary skin. Unscheduled DNA synthesis appeared depressed in actinic keratosis patients, as compared with controls. In all examined subjects, however, cell strains from exposed skin showed a DNA repair more active than cell strains from unexposed skin. These findings show that skin cancer may be promoted in actinic keratosis patients by a defect of DNA repair. The exalted DNA repair of chronically sun exposed skin is probably the consequence of a defensive process caused by enzymatic induction. (orig.) [de

  2. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

    Science.gov (United States)

    Cox, Annie M; Goodwin, Kelly D

    2013-08-15

    The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. Published by Elsevier Ltd.

  3. Physicians' current practices and opportunities for DNA banking of dying patients with cancer.

    Science.gov (United States)

    Quillin, John M; Bodurtha, Joann N; Siminoff, Laura A; Smith, Thomas J

    2011-05-01

    The availability of genetic tests for cancer susceptibility is increasing. Current tests, however, have limited clinical sensitivity. Even when clinically valid tests are available, the genetic counseling and informed consent process might not be feasible for dying patients with cancer. DNA banking preserves the opportunity for future research or clinical testing and may provide critical opportunities for surviving relatives. This study explored the current practices and potential for DNA banking for cancer susceptibility among oncologists specializing in palliative care. Palliative care oncologists actively providing clinical care for dying patients with cancer were recruited for an online survey. Descriptive statistics for DNA banking practices, perceived qualification to recommend banking, and potential predictors were assessed. Data were collected from 49 physicians (37% recruitment rate). Eighty percent reported assessing at least some patients for genetic cancer susceptibility in the past 12 months. No participants reported banking DNA for patients in the past 12 months. Only 5% reported feeling at least somewhat qualified to order DNA banking. A Web-based risk assessment tool and genetic counselor on staff were perceived as the most helpful potential resources. Despite its potential, DNA banking is not being used by palliative care oncologists.

  4. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR.

    Science.gov (United States)

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland-Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). 2-LTR circles quantification in HIV-infected patients proved to be more

  5. Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection.

    Directory of Open Access Journals (Sweden)

    Tran Thi Ngoc Ha

    Full Text Available BACKGROUND: Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue. METHODS AND FINDINGS: A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome. CONCLUSIONS: Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.

  6. Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

    Science.gov (United States)

    de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

    2017-08-01

    The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

  7. Plasma cell-free DNA and its DNA integrity as biomarker to distinguish prostate cancer from benign prostatic hyperplasia in patients with increased serum prostate-specific antigen.

    Science.gov (United States)

    Feng, Jiang; Gang, Feng; Li, Xiao; Jin, Tang; Houbao, Huang; Yu, Cao; Guorong, Li

    2013-08-01

    To investigate whether plasma cell-free DNA (cfDNA) or its integrity could differentiate prostate cancer from benign prostate hyperplasia (BPH) in patients with serum prostate-specific antigen (PSA) ≥ 4 ng/ml. Ninety-six patients with prostate cancer and 112 patients with BPH were enrolled. cfDNA levels in plasma before prostate biopsy were quantified by real-time PCR amplification of ALU gene (product size of 115 bp), and quantitative ratio of ALU (247 bp) to ALU (115 bp) reflected the integrity of cfDNA. In patients with serum PSA ≥ 4 ng/ml, there were significant differences in plasma cfDNA or its integrity between the patients with prostate cancer (19.74 ± 4.43, 0.34 ± 0.05) and patients with BPH (7.36 ± 1.58, 0.19 ± 0.03; P Prostate cancer could be differentiated with a sensitivity of 73.2 % and a specificity of 72.7 % by cfDNA (AUC = 0.864). The integrity of cfDNA had a sensitivity of 81.7 % and a specificity of 78.8 % for the distinguishing prostate cancer from BPH (AUC = 0.910). cfDNA and its integrity could be applied to differentiate prostate cancer from BPH in patients with serum PSA ≥ 4 ng/ml.

  8. Human herpesvirus infections of the central nervous system: laboratory diagnosis based on DNA detection by nested PCR in plasma and cerebrospinal fluid samples.

    Science.gov (United States)

    Rimério, Carla Aparecida Tavares; De Oliveira, Renato Souza; de Almeida Bonatelli, Murilo Queiroz; Nucci, Anamarli; Costa, Sandra Cecília Botelho; Bonon, Sandra Helena Alves

    2015-04-01

    Infections of the central nervous systems (CNS) present a diagnostic problem for which an accurate laboratory diagnosis is essential. Invasive practices, such as cerebral biopsy, have been replaced by obtaining a polymerase chain reaction (PCR) diagnosis using cerebral spinal fluid (CSF) as a reference method. Tests on DNA extracted from plasma are noninvasive, thus avoiding all of the collateral effects and patient risks associated with CSF collection. This study aimed to determine whether plasma can replace CSF in nested PCR analysis for the detection of CNS human herpesvirus (HHV) diseases by analysing the proportion of patients whose CSF nested PCR results were positive for CNS HHV who also had the same organism identified by plasma nested PCR. In this study, CSF DNA was used as the "gold standard," and nested PCR was performed on both types of samples. Fifty-two patients with symptoms of nervous system infection were submitted to CSF and blood collection. For the eight HHV, one positive DNA result-in plasma and/or CSF nested PCR-was considered an active HHV infection, whereas the occurrence of two or more HHVs in the same sample was considered a coinfection. HHV infections were positively detected in 27/52 (51.9%) of the CSF and in 32/52 (61.5%) of the plasma, difference not significant, thus nested PCR can be performed on plasma instead of CSF. In conclusion, this findings suggest that plasma as a useful material for the diagnosis of cases where there is any difficulty to perform a CSF puncture. © 2015 Wiley Periodicals, Inc.

  9. Repurposing environmental DNA samples: Detecting the western pearlshell (Margaritifera falcata) as a proof of concept

    Science.gov (United States)

    Joseph C. Dysthe; Torrey Rodgers; Thomas W. Franklin; Kellie J. Carim; Michael K. Young; Kevin S. McKelvey; Karen E. Mock; Michael K. Schwartz

    2018-01-01

    Information on the distribution of multiple species in a common landscape is fundamental to effective conservation and management. However, distribution data are expensive to obtain and often limited to high-profile species in a system. A recently developed technique, environmental DNA (eDNA) sampling, has been shown to be more sensitive than traditional detection...

  10. Diverse replication-associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats.

    Science.gov (United States)

    Kemenesi, Gábor; Kurucz, Kornélia; Zana, Brigitta; Földes, Fanni; Urbán, Péter; Vlaschenko, Anton; Kravchenko, Kseniia; Budinski, Ivana; Szodoray-Parádi, Farkas; Bücs, Szilárd; Jére, Csaba; Csősz, István; Szodoray-Parádi, Abigél; Estók, Péter; Görföl, Tamás; Boldogh, Sándor; Jakab, Ferenc

    2018-03-01

    Circular replication-associated protein encoding single-stranded DNA (CRESS DNA) viruses are increasingly recognized worldwide in a variety of samples. Representative members include well-described veterinary pathogens with worldwide distribution, such as porcine circoviruses or beak and feather disease virus. In addition, numerous novel viruses belonging to the family Circoviridae with unverified pathogenic roles have been discovered in different human samples. Viruses of the family Genomoviridae have also been described as being highly abundant in different faecal and environmental samples, with case reports showing them to be suspected pathogens in human infections. In order to investigate the genetic diversity of these viruses in European bat populations, we tested guano samples from Georgia, Hungary, Romania, Serbia and Ukraine. This resulted in the detection of six novel members of the family Circoviridae and two novel members of the family Genomoviridae. Interestingly, a gemini-like virus, namely niminivirus, which was originally found in raw sewage samples in Nigeria, was also detected in our samples. We analyzed the nucleotide composition of members of the family Circoviridae to determine the possible host origins of these viruses. This study provides the first dataset on CRESS DNA viruses of European bats, and members of several novel viral species were discovered.

  11. Investigations on DNA repair in peripheric lymphocytes of arthritic patients treated at Badgastein

    International Nuclear Information System (INIS)

    Egg, D.; Guenther, R.; Klein, W.; Kocsis, F.; Altmann, H.

    1976-01-01

    The DNA repair capacity in peripheric lymphocytes was studied in 18 arthritic patients after completion of a therapy at Badgastein. It was found that excision repair determined by the ''student test'' was significantly increased for 11 patients as compared to the level before treatment. In 4 patients no significant change was found. A clear decrease of DNA excision repair was encountered in 2 patients. One patient showed a complete inhibition of DNA excision repair before as well as after the treatment. The role of different parameters such as environmental radiaton exposure, altitude, ambient temperature for the observed changes cannot be deduced from the results obtained as yet and shall be clarified in subsequent investigation. (G.G.)

  12. Relationship between DNA replication and DNA repair in human lymphocytes proliferating in vitro in the presence and in absence of mutagen

    International Nuclear Information System (INIS)

    Szyfter, K.; Wielgosz, M.Sz.; Kujawski, M.; Jaloszynski, P.; Zajaczek, S.

    1995-01-01

    The effects of mutagens on DNA replication and DNA repair were studied in peripheral blood lymphocytes (PBL) obtained from 21 healthy subjects, 2 samples from healthy heterozygote of ''Xeroderma pigmentosum'' (XP) and 2 samples from patient with clinically recognised XP. Inter-individual variations were found in DNA replication and in the level of spontaneous DNA repair measured under standard culture condition. Exposure of human PBL proliferating in vitro to B(a)P was followed by a partial inhibition of replicative DNA synthesis in all subjects and by an induction of DNA repair in healthy subjects. In XP patients DNA repair synthesis remained at the level attributed to spontaneous DNA repair. The response to mutagen varied individually. Results were analysed statistically. It was established that the studied indices of DNA synthesis correlate well with each other. The highest correlation was found between the levels of spontaneous and B(a)P-induced DNA repair. It is concluded that the level of spontaneous DNA repair is predictive for an estimation of cells ability to repair DNA damage. Inter-individual variations in the inhibition of DNA replication and in DNA repair synthesis are also dependent on the type of mutagen as shown by effects of other mutagens. Different effects of mutagen exposure on the inhibition of DNA replicative synthesis and induction of DNA repair can be explained by genetically controlled differences in the activity of enzymes responsible for mutagen processing and lesion removal. (author). 37 refs, 2 figs, 2 tabs

  13. Salivary DNA and markers of oxidative stress in patients with chronic periodontitis.

    Science.gov (United States)

    Baňasová, Lenka; Kamodyová, Natália; Janšáková, Katarína; Tóthová, Ľubomíra; Stanko, Peter; Turňa, Ján; Celec, Peter

    2015-03-01

    Previous observational studies have shown that periodontal status is associated with salivary markers of oxidative damage. A direct comparison of periodontitis patients and controls using a wide palette of salivary markers of oxidative stress is lacking. Characteristics of salivary DNA in periodontitis are unknown. The aim of this study was to compare the salivary markers of oxidative stress and characteristics of salivary DNA between patients with chronic periodontitis and periodontitis-free controls. Saliva was collected from 23 patients with chronic periodontitis and 19 periodontitis-free controls. All participants underwent a clinical periodontal examination. Markers of oxidative and carbonyl stress were measured in saliva. Human and bacterial DNA was quantified, and human DNA integrity was assessed. Salivary thiobarbituric acid-reacting substances were higher in patients than in controls; at least in men, the difference was significant (p periodontitis patients. The results confirmed the association of salivary thiobarbituric acid-reacting substances with periodontitis. Lipid peroxidation in periodontitis seems to be caused by increased production of reactive oxygen species in men and by decreased antioxidant status in women. Whether lower salivary DNA integrity is involved in the pathogenesis of periodontitis remains to be elucidated. Salivary thiobarbituric acid-reacting substances are associated with periodontitis at least on a population level. Sex-specific causes of lipid peroxidation might point towards different pathogenic mechanisms.

  14. Detecção de DNA de Leishmania braziliensis em pacientes de leishmaniose tegumentar americana Detección de DNA de Leishmania braziliensis en pacientes de leishmaniose tegumentaria americana Detection of Leishmania braziliensis DNA in American tegumentary leishmaniasis patients

    Directory of Open Access Journals (Sweden)

    Leila Martins

    2010-06-01

    Full Text Available Foi realizado diagnóstico para leishmaniose tegumentar americana a partir de sangue de pacientes residentes em dois municípios endêmicos do estado de Pernambuco. O DNA de 119 amostras de sangue foi extraído e submetido a reação em cadeia da polimerase. Utilizaram-se primers do minicírculo do DNA do cinetoplasto (kDNA de Leishmania braziliensis, circulante em Pernambuco, cuja seqüência-alvo gera um fragmento de 750 pares de bases. No total 58 (48,7% indivíduos apresentaram amplificação positiva e 61 (51,3% negativa. Das amostras positivas para a PCR, 37 (≅ 64% pertenciam a indivíduos tratados e sem lesão. Conclui-se que a técnica de PCR é eficaz para identificar o DNA de leishmânia em material de biópsias e em sangue venoso.Fue realizado diagnóstico para leishmaniosis tegumentaria americana a partir de sangre de pacientes residentes en dos municipios endémicos del estado de Pernambuco (Noreste de Brasil. El DNA de 119 muestras de sangre fue extraído y sometido a la reacción en cadena de la polimerasa. Se utilizaron primers del minicírculo del DNA del cinetoplasto (kDNA de Leishmania braziliensis, circulante en Pernambuco, cuya secuencia blanco genera un fragmento de 750 pares de bases. En total 58 (48,7% individuos presentaron amplificación positiva y 61 (51,3% negativa. De las muestras positivas para la PCR, 37 (≅64% pertenecían a individuos tratados y sin lesión. Se concluyó que la técnica de la PCR es eficaz para identificar el DNA de Leishmania en material de biopsias y en sangre venosa.Diagnostic tests for American tegumentary leishmaniasis were performed on blood samples of patients living in two endemic municipalities in the state of Pernambuco, Northeastern Brazil. DNA was extracted from 119 samples and used as template for polymerase chain reaction (PCR analysis. The tests used primers specific for the kinetoplast mini-circle DNA (kDNA of Leishmania braziliensis, a species circulating in Pernambuco, which

  15. Quantification of Cell-Free HER-2 DNA in Plasma from Breast Cancer Patients

    DEFF Research Database (Denmark)

    Sørensen, Patricia Diana; Andersen, Rikke Fredslund; Pallisgaard, Niels

    2015-01-01

    The purpose of this study was to quantify the free-circulating plasma HER-2 DNA (cfHER-2 DNA) and to assess the ability of analysis to discriminate between patients with primary breast cancer and healthy controls in order to detect metastatic recurrence in comparison with serum HER-2 protein...... and also HER-2 gene amplification. The study population consisted of 100 patients with primary breast cancer and 50 healthy female donors. An additional 22 patients with metastases were subsequently included. cfHER-2 DNA was quantified with a quantitative PCR method together with a reference gene. RESULTS......: Using a cut-off of 2.5 for the ratio of the cfHER-2 DNA/reference gene, three (of 15) tissue HER-2-positive patients had a ratio >2.5 prior to the detection of metastatic disease. In the post-metastatic/pre-chemotherapy setting, 11 (of 23) tissue HER-2-positive patients with metastases had a ratio >2...

  16. Ionizing radiation-induced DNA injury and damage detection in patients with breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Borrego-Soto, Gissela; Ortiz-Lopez, Rocio; Rojas-Martinez, Augusto, E-mail: arojasmtz@gmail.com, E-mail: augusto.rojasm@uanl.mx [Departamento de Bioquímica y Medicina Molecular, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León (Mexico)

    2015-10-15

    Breast cancer is the most common malignancy in women. Radiotherapy is frequently used in patients with breast cancer, but some patients may be more susceptible to ionizing radiation, and increased exposure to radiation sources may be associated to radiation adverse events. This susceptibility may be related to deficiencies in DNA repair mechanisms that are activated after cell-radiation, which causes DNA damage, particularly DNA double strand breaks. Some of these genetic susceptibilities in DNA-repair mechanisms are implicated in the etiology of hereditary breast/ovarian cancer (pathologic mutations in the BRCA 1 and 2 genes), but other less penetrant variants in genes involved in sporadic breast cancer have been described. These same genetic susceptibilities may be involved in negative radiotherapeutic outcomes. For these reasons, it is necessary to implement methods for detecting patients who are susceptible to radiotherapy-related adverse events. This review discusses mechanisms of DNA damage and repair, genes related to these functions, and the diagnosis methods designed and under research for detection of breast cancer patients with increased radiosensitivity. (author)

  17. Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success

    Science.gov (United States)

    Maxwell, Elisabeth A.; Marshall, Andrea D.; Christensen, Ana B.

    2015-01-01

    Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing. PMID:26413431

  18. The effect of DNA degradation bias in passive sampling devices on metabarcoding studies of arthropod communities and their associated microbiota.

    Science.gov (United States)

    Krehenwinkel, Henrik; Fong, Marisa; Kennedy, Susan; Huang, Edward Greg; Noriyuki, Suzuki; Cayetano, Luis; Gillespie, Rosemary

    2018-01-01

    PCR amplification bias is a well-known problem in metagenomic analysis of arthropod communities. In contrast, variation of DNA degradation rates is a largely neglected source of bias. Differential degradation of DNA molecules could cause underrepresentation of taxa in a community sequencing sample. Arthropods are often collected by passive sampling devices, like malaise traps. Specimens in such a trap are exposed to varying periods of suboptimal storage and possibly different rates of DNA degradation. Degradation bias could thus be a significant issue, skewing diversity estimates. Here, we estimate the effect of differential DNA degradation on the recovery of community diversity of Hawaiian arthropods and their associated microbiota. We use a simple DNA size selection protocol to test for degradation bias in mock communities, as well as passively collected samples from actual Malaise traps. We compare the effect of DNA degradation to that of varying PCR conditions, including primer choice, annealing temperature and cycle number. Our results show that DNA degradation does indeed bias community analyses. However, the effect of this bias is of minor importance compared to that induced by changes in PCR conditions. Analyses of the macro and microbiome from passively collected arthropod samples are thus well worth pursuing.

  19. Increased cell-free DNA concentrations in patients with obstructive sleep apnea.

    Science.gov (United States)

    Shin, Chol; Kim, Jin K; Kim, Je H; Jung, Ki H; Cho, Kyung J; Lee, Chang K; Lee, Seung G

    2008-12-01

    Blood concentrations of cell-free DNA, which is considered to be released during apoptosis, are elevated under some pathological conditions such as cardiovascular disease and cancer. The association between obstructive sleep apnea (OSA) and cell-free DNA concentrations has not been reported so far. The purpose of the present study was to examine the association between OSA and plasma DNA concentrations. A case-control study was conducted using a total of 164 men aged 39-67 years, who were free of coronary heart disease and cancer. Laboratory-based overnight polysomnography was performed for all participants. On the basis of polysomnography, patients with an apnea-hypopnea index (AHI) = 5-30 events/h were defined as having mild-moderate OSA (n = 33) and those with >30 events/h were defined as having severe OSA (n = 49). All 82 controls had AHI DNA concentrations from all participants were analyzed for the beta-globin gene using fluorescence-based real-time polymerase chain reaction. Patients with severe OSA had significantly higher plasma DNA concentrations than persons with mild-moderate OSA and those without OSA (P DNA concentration (P DNA concentrations (>8 microg/L) had approximately fourfold higher odds of OSA than those with low DNA levels. Further data are warranted to confirm the association for men and to evaluate the association for women.

  20. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors

    International Nuclear Information System (INIS)

    Cox, Annie M.; Goodwin, Kelly D.

    2013-01-01

    Highlights: • DNA extraction methods affected measured qPCR target recovery. • Recovery and variability differed, sometimes by more than an order of magnitude. • SCODA did not offer significant improvement with PCR-inhibited seawater. • Aggressive lysis did appear to improve target recovery. • Reliable and affordable correction methods are needed for quantitative PCR. -- Abstract: The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications

  1. A DNA based method to detect the grapevine root-rotting fungus Roesleria subterranea in soil and root samples

    Directory of Open Access Journals (Sweden)

    S. Neuhauser

    2009-05-01

    Full Text Available Roesleria subterranea causes root rot in grapevine and fruit trees. The fungus has long been underestimated as a weak parasite, but during the last years it has been reported to cause severe damages in German vineyards. Direct, observation-based detection of the parasite is time consuming and destructive, as large parts of the rootstocks have to be uprooted and screened for the tiny, stipitate, hypogeous ascomata of R. subterranea. To facilitate rapid detection in vineyards, protocols to extract DNA from soil samples and grapevine roots, and R.-subterranea-specific PCR primers were designed. Twelve DNA-extraction protocols for soil samples were tested in small-scale experiments, and selected parameters were optimised. A protocol based on ball-mill homogenization, DNA extraction with SDS, skim milk, chloroform, and isopropanol, and subsequent purifi cation of the raw extracts with PVPP-spin-columns was most effective. This DNA extraction protocol was found to be suitable for a wide range of soil-types including clay, loam and humic-rich soils. For DNA extraction from grapevine roots a CTAB-based protocol was more reliable for various grapevine rootstock varieties. Roesleria-subterranea-specific primers for the ITS1-5.8S-ITS2 rDNA region were developed and tested for their specifi city to DNA extracts from eleven R. subterranea strains isolated from grapevine and fruit trees. No cross reactions were detected with DNA extracts from 44 different species of fungi isolated from vineyard soils. The sensitivity of the species-specifi c primers in combination with the DNA extraction method for soil was high: as little as 100 fg μl-1 R.-subterranea-DNA was suffi cient for a detection in soil samples and plant material. Given that specifi c primers are available, the presented method will also allow quick and large-scale testing for other root pathogens.

  2. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

    International Nuclear Information System (INIS)

    Martínez-Galán, Joaquina; Ríos, Sandra; Delgado, Juan Ramón; Torres-Torres, Blanca; Núñez, María Isabel; López-Peñalver, Jesús; Del Moral, Rosario; Ruiz De Almodóvar, José Mariano; Menjón, Salomón; Concha, Ángel; Chamorro, Clara

    2014-01-01

    Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment

  3. Applicability of Ion Torrent Colon and Lung sequencing panel on circulating cell-free DNA

    DEFF Research Database (Denmark)

    Demuth, Christina; Tranberg Madsen, Anne; Larsen, Anne Winther

    of targeted sequencing have been optimised for clinical use on FFPE, e.g. the Ion Torrent Colon and Lung panel. The size of DNA extracted from FFPE tissue is comparable with that from cfDNA. We therefore investigated the performance of the clinically relevant Ion Torrent Colon and Lung panel on cfDNA. Methods...... a baseline for the panel. Lastly, the panel was tested on 52 patient samples. Patient plasma samples are from a previously collected cohort of EGFR wild-type non-small cell lung cancer patients (ClinicalTrial.gov: NCT02043002) All samples were sequenced using the Ion Torrent Oncomine Solid Tumor DNA kit...... (Colon and Lung panel) from Thermo Fisher. Sample preparation was performed using the Ion Torrent Chef and sequencing was performed on the Personal Genome Machine (PGM) system. Data was analyzed using the Torrent Suite software, and variants called by Ion Reporter. Results: No somatic mutations were...

  4. Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

    Science.gov (United States)

    Molecular methods, such as DNA barcoding, have the potential in enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biom...

  5. Fluorescence quantitative PCR in detection of HBV DNA

    International Nuclear Information System (INIS)

    Shen Zheng; Li Ming; Shen Xia

    2003-01-01

    Objective: To study the relationship between the serum content of HBV-DNA and expression of serological markers with HBV infection patients. Methods: Serum samples from 375 hepatitis B patients with different clinical status and 70 normal persons were quantitated for HBV-DNA by FQ-PCR. Results: The average of HBV-DNA contents in the patient in the groups of HBsAg (+) and of HBeAg(+) were significantly higher than those in the group of HBsAg(-) and of HBeAg(-). Even in the group of HBeAg negative, high HBV-DNA contents might still be present in both the HBeAg(+) and HBeAg(-) groups. Conclusion: FQ-PCR can be used to monitor the real state of HBV infection, replication and the course of disease

  6. Improved detection of endoparasite DNA in soil sample PCR by the use of anti-inhibitory substances.

    Science.gov (United States)

    Krämer, F; Vollrath, T; Schnieder, T; Epe, C

    2002-09-26

    Although there have been numerous microbial examinations of soil for the presence of human pathogenic developmental parasite stages of Ancylostoma caninum and Toxocara canis, molecular techniques (e.g. DNA extraction, purification and subsequent PCR) have scarcely been applied. Here, DNA preparations of soil samples artificially contaminated with genomic DNA or parasite eggs were examined by PCR. A. caninum and T. canis-specific primers based on the ITS-2 sequence were used for amplification. After the sheer DNA preparation a high content of PCR-interfering substances was still detectable. Subsequently, two different inhibitors of PCR-interfering agents (GeneReleaser, Bioventures Inc. and Maximator, Connex GmbH) were compared in PCR. Both substances increased PCR sensitivity greatly. However, comparison of the increase in sensitivity achieved with the two compounds demonstrated the superiority of Maximator, which enhanced sensitivity to the point of permitting positive detection of a single A. caninum egg and three T. canis eggs in a soil sample. This degree of sensitivity could not be achieved with GeneReleaser for either parasite Furthermore, Maximator not only increased sensitivity; it also cost less, required less time and had a lower risk of contamination. Future applications of molecular methods in epidemiological examinations of soil samples are discussed/elaborated.

  7. [The structure of the leukocyte DNA in leukemia patients during chemotherapy].

    Science.gov (United States)

    Tarakanov, M P; Moskaleva, E Iu; Bezobrazova, L V; Semenova, O I; Korneva, E N; Telegin, L Iu

    1993-01-01

    The authors studied the degree of DNA damage in in vitro cultured human peripheral lymphocytes (PL) and Jurcat's human T-cell lymphoma cells exposed to a stabilized 4 OH-cyclophosphan-mamophosphatide (MA) derivative, as well as in the leukocytes from patients with leukemia who were treated with cyclophosphan. There was an increase in alkaline DNA denaturation rate of LP lysates and T-cell lymphoma cells, which was in proportion to MA concentrations, and a higher sensitivity of LP to the genotoxic effect of MA given in doses of 5-10 micrograms/ml than that of Jurcat's cells, as well as high peripheral lymphocyte and neutrophil DNA damages in patients with leukemia during chemotherapy. The authors consider that the accumulation of single-strand breaks and alkaline-labile sites, which was recorded from the increase in alkaline DNA denaturation rate of cell lysates, is a highly sensitive test for detecting DNA damages in resting and slowly proliferating cells and can be useful in revealing and evaluating the severity of human genotoxic effects.

  8. DNA damage focus analysis in blood samples of minipigs reveals acute partial body irradiation.

    Directory of Open Access Journals (Sweden)

    Andreas Lamkowski

    Full Text Available Radiation accidents frequently involve acute high dose partial body irradiation leading to victims with radiation sickness and cutaneous radiation syndrome that implements radiation-induced cell death. Cells that are not lethally hit seek to repair ionizing radiation (IR induced damage, albeit at the expense of an increased risk of mutation and tumor formation due to misrepair of IR-induced DNA double strand breaks (DSBs. The response to DNA damage includes phosphorylation of histone H2AX in the vicinity of DSBs, creating foci in the nucleus whose enumeration can serve as a radiation biodosimeter. Here, we investigated γH2AX and DNA repair foci in peripheral blood lymphocytes of Göttingen minipigs that experienced acute partial body irradiation (PBI with 49 Gy (± 6% Co-60 γ-rays of the upper lumbar region. Blood samples taken 4, 24 and 168 hours post PBI were subjected to γ-H2AX, 53BP1 and MRE11 focus enumeration. Peripheral blood lymphocytes (PBL of 49 Gy partial body irradiated minipigs were found to display 1-8 DNA damage foci/cell. These PBL values significantly deceed the high foci numbers observed in keratinocyte nuclei of the directly γ-irradiated minipig skin regions, indicating a limited resident time of PBL in the exposed tissue volume. Nonetheless, PBL samples obtained 4 h post IR in average contained 2.2% of cells displaying a pan-γH2AX signal, suggesting that these received a higher IR dose. Moreover, dispersion analysis indicated partial body irradiation for all 13 minipigs at 4 h post IR. While dose reconstruction using γH2AX DNA repair foci in lymphocytes after in vivo PBI represents a challenge, the DNA damage focus assay may serve as a rapid, first line indicator of radiation exposure. The occurrence of PBLs with pan-γH2AX staining and of cells with relatively high foci numbers that skew a Poisson distribution may be taken as indicator of acute high dose partial body irradiation, particularly when samples are available

  9. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    Energy Technology Data Exchange (ETDEWEB)

    Ali, M E; Hashim, U [Institute of Nano Electronic Engineering (INNE), Universiti Malaysia Perlis, Lot 104-108, Tingkat 1, Block A, Taman Pertiwi Indah, Jalan Kangar-Alor Star, Seriab, 01000 Kangar, Perlis (Malaysia); Mustafa, S; Che Man, Y B; Yusop, M H M [Halal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Bari, M F [School of Materials Engineering, University Malaysia Perlis, Seriab 01000, Kangar, Perlis (Malaysia); Islam, Kh N [Department of Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia); Hasan, M F, E-mail: uda@unimap.edu.my [Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia)

    2011-05-13

    We used 40 {+-} 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 {sup 0}C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 {mu}g ml{sup -1} swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  10. Nanoparticle sensor for label free detection of swine DNA in mixed biological samples

    International Nuclear Information System (INIS)

    Ali, M E; Hashim, U; Mustafa, S; Che Man, Y B; Yusop, M H M; Bari, M F; Islam, Kh N; Hasan, M F

    2011-01-01

    We used 40 ± 5 nm gold nanoparticles (GNPs) as colorimetric sensor to visually detect swine-specific conserved sequence and nucleotide mismatch in PCR-amplified and non-amplified mitochondrial DNA mixtures to authenticate species. Colloidal GNPs changed color from pinkish-red to gray-purple in 2 mM PBS. Visually observed results were clearly reflected by the dramatic reduction of surface plasmon resonance peak at 530 nm and the appearance of new features in the 620-800 nm regions in their absorption spectra. The particles were stabilized against salt-induced aggregation upon the adsorption of single-stranded DNA. The PCR products, without any additional processing, were hybridized with a 17-base probe prior to exposure to GNPs. At a critical annealing temperature (55 0 C) that differentiated matched and mismatched base pairing, the probe was hybridized to pig PCR product and dehybridized from the deer product. The dehybridized probe stuck to GNPs to prevent them from salt-induced aggregation and retained their characteristic red color. Hybridization of a 27-nucleotide probe to swine mitochondrial DNA identified them in pork-venison, pork-shad and venison-shad binary admixtures, eliminating the need of PCR amplification. Thus the assay was applied to authenticate species both in PCR-amplified and non-amplified heterogeneous biological samples. The results were determined visually and validated by absorption spectroscopy. The entire assay (hybridization plus visual detection) was performed in less than 10 min. The LOD (for genomic DNA) of the assay was 6 μg ml -1 swine DNA in mixed meat samples. We believe the assay can be applied for species assignment in food analysis, mismatch detection in genetic screening and homology studies between closely related species.

  11. Evaluating manta ray mucus as an alternative DNA source for population genetics study: underwater-sampling, dry-storage and PCR success

    Directory of Open Access Journals (Sweden)

    Tom Kashiwagi

    2015-08-01

    Full Text Available Sharks and rays are increasingly being identified as high-risk species for extinction, prompting urgent assessments of their local or regional populations. Advanced genetic analyses can contribute relevant information on effective population size and connectivity among populations although acquiring sufficient regional sample sizes can be challenging. DNA is typically amplified from tissue samples which are collected by hand spears with modified biopsy punch tips. This technique is not always popular due mainly to a perception that invasive sampling might harm the rays, change their behaviour, or have a negative impact on tourism. To explore alternative methods, we evaluated the yields and PCR success of DNA template prepared from the manta ray mucus collected underwater and captured and stored on a Whatman FTA™ Elute card. The pilot study demonstrated that mucus can be effectively collected underwater using toothbrush. DNA stored on cards was found to be reliable for PCR-based population genetics studies. We successfully amplified mtDNA ND5, nuclear DNA RAG1, and microsatellite loci for all samples and confirmed sequences and genotypes being those of target species. As the yields of DNA with the tested method were low, further improvements are desirable for assays that may require larger amounts of DNA, such as population genomic studies using emerging next-gen sequencing.

  12. Detection of free circulating Epstein-Barr virus DNA in plasma of patients with Hodgkin’s disease

    Directory of Open Access Journals (Sweden)

    Juliane Garcez Musacchio

    Full Text Available CONTEXT AND OBJECTIVE: Free circulating Epstein-Barr virus (EBV DNA is often present in the plasma of Hodgkin’s disease patients. The aim here was to evaluate the prevalence of this finding, its correlation with the immunohistochemical expression of LMP-1 (latent membrane protein 1 and the influence of other clinical factors. DESIGN AND SETTING: Prospective study in two public tertiary institutions: Hematology Service, Universidade Federal do Rio de Janeiro, and Oncology Service, Instituto Nacional do Câncer, Rio de Janeiro. METHODS: A cohort of 30 patients with newly diagnosed Hodgkin’s disease was studied. The control group consisted of 13 healthy adult volunteers. EBV DNA was determined by conventional polymerase chain reaction (PCR. RESULTS: The median age was 28 years, and 16 patients were women. Advanced disease was present in 19 patients, and six were HIV-positive. EBV DNA was present in the plasma of 13 patients and one control (43% versus 8%, p = 0.03. EBV DNA prevalence was higher in HIV-positive patients (100% versus 29%, p = 0.0007 and those with advanced disease (63% versus 9%, p = 0.006. Among HIV-negative patients alone, EBV DNA prevalence remained higher in those with advanced disease. EBV DNA was found in 10/11 patients with LMP-1 expression in the lymph nodes, and in 3/19 without LMP-1 expression (kappa coefficient = 0.72. CONCLUSION: EBV DNA was present in 91% of patients with EBV-associated Hodgkin’s disease, and in all patients with HIV-associated Hodgkin’s disease. EBV DNA prevalence was higher in patients with advanced disease, irrespective of HIV status.

  13. Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring.

    Science.gov (United States)

    Perets, Ruth; Greenberg, Orli; Shentzer, Talia; Semenisty, Valeria; Epelbaum, Ron; Bick, Tova; Sarji, Shada; Ben-Izhak, Ofer; Sabo, Edmond; Hershkovitz, Dov

    2018-05-01

    Many new pancreatic cancer treatment combinations have been discovered in recent years, yet the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains grim. The advent of new treatments highlights the need for better monitoring tools for treatment response, to allow a timely switch between different therapeutic regimens. Circulating tumor DNA (ctDNA) is a tool for cancer detection and characterization with growing clinical use. However, currently, ctDNA is not used for monitoring treatment response. The high prevalence of KRAS hotspot mutations in PDAC suggests that mutant KRAS can be an efficient ctDNA marker for PDAC monitoring. Seventeen metastatic PDAC patients were recruited and serial plasma samples were collected. CtDNA was extracted from the plasma, and KRAS mutation analysis was performed using next-generation sequencing and correlated with serum CA19-9 levels, imaging, and survival. Plasma KRAS mutations were detected in 5/17 (29.4%) patients. KRAS ctDNA detection was associated with shorter survival (8 vs. 37.5 months). Our results show that, in ctDNA positive patients, ctDNA is at least comparable to CA19-9 as a marker for monitoring treatment response. Furthermore, the rate of ctDNA change was inversely correlated with survival. Our results confirm that mutant KRAS ctDNA detection in metastatic PDAC patients is a poor prognostic marker. Additionally, we were able to show that mutant KRAS ctDNA analysis can be used to monitor treatment response in PDAC patients and that ctDNA dynamics is associated with survival. We suggest that ctDNA analysis in metastatic PDAC patients is a readily available tool for disease monitoring. Avoiding futile chemotherapy in metastatic pancreatic ductal adenocarcinoma (PDAC) patients by monitoring response to treatment is of utmost importance. A novel biomarker for monitoring treatment response in PDAC, using mutant KRAS circulating tumor DNA (ctDNA), is proposed. Results, although limited by small sample numbers

  14. Measurement of indicator genes using global complementary DNA (cDNA) amplification, by polyadenylic acid reverse transcriptase polymerase chain reaction (poly A RT-PCR): A feasibility study using paired samples from tissue and ductal juice in patients undergoing pancreatoduodenectomy.

    Science.gov (United States)

    Sanyal, Sudip; Siriwardena, Ajith K; Byers, Richard

    2018-06-01

    The aim of this study is to compare gene expression profiles in RNA isolated from pancreatic ductal juice with the RNA expression profiles of the same genes from matched intra-operative tissue samples from pancreatic tumours. Intra-operative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreatoduodenectomy for clinically suspected pancreatic cancer and a precursor lesion, main-duct intraductal papillary mucinous neoplasm. RNA was isolated and Poly A PCR was used to globally amplify the RNA. Real-time polymerase chain reaction (RT-PCR) was used to measure expression levels of 17 genes selected from microarray studies. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. The study was approved by Regional Ethics Committee. Mesothelin (MSLN) showed significant correlation (p cDNA using poly A PCR is technically feasible. Application of the technique to non-invasively obtained pancreatic juice during endoscopic assessment of tumours and the use of gene arrays of cancer indicator genes are the next steps in development of this technique. Copyright © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.

  15. Clinical significance of changes of expression of anti-dsDNA antibody in serum in patients with SLE

    International Nuclear Information System (INIS)

    Chen Xingguo; Zhang Xiaoli; Liu Chunyan; Cao Jiwei; Du Tongxing; Wang Zizheng

    2008-01-01

    Objective: To investigate the significance of anti-dsDNA antibody in diagnosis and treatment of SLE through measurement of changes of serum anti-dsDNA antibody expression in patients with SLE. Methods: Serum anti-dsDNA antibody was detected with radioisotope method in 60 patients with SLE and 33 controls (consisted of patients with other collagen diseases including Sjogren syndrome, systemic sclerosis, rheumatoid arthritis, polymyositis, mixed connective tissue disease, ankylosing spondylitis). Clinical manifestation and laboratory findings in the SLE patients were studied in detail. Results: (1) Serum anti-dsDNA antibody was positive in 39 of the 60 SLE patients with only two false positive cases in the 33 controls: a sensitivity of 65% and specificity of 93. 3%. (2) In SLE patients, positivity of anti-dsDNA antibody was not correlated with positivity of anti-Sm antibody (P>0.05), but was correlated with positivity of anti-SSA antibody (P<0.05). (3) Incidences of alopecia, skin rashes, oral mucosal ulcer, proteinuria were significantly higher in SLE patients with positive anti-dsDNA antibody than those in SLE patients with negative anti-dsDNA antibody (P<0.05). (4) Incidences of leukopenia and thrombocytopenia were also significantly higher in SLE patients with positive anti-dsDNA antibody (P<0.05). Conclusion: Anti-dsDNA antibody could be taken as a specific marker of SLE and the serum expression were positively correlated with the activity and severity of the disease. (authors)

  16. TH-C-18A-09: Exam and Patient Parameters Affecting the DNA Damage Response Following CT Studies

    International Nuclear Information System (INIS)

    Elgart, S; Adibi, A; Bostani, M; Ruehm, S; Enzmann, D; McNitt-Gray, M; Iwamoto, K

    2014-01-01

    Purpose: To identify exam and patient parameters affecting the biological response to CT studies using in vivo and ex vivo blood samples. Methods: Blood samples were collected under IRB approval from 16 patients undergoing clinically-indicated CT exams. Blood was procured prior to, immediately after and 30minutes following irradiation. A sample of preexam blood was placed on the patient within the exam region for ex vivo analysis. Whole blood samples were fixed immediately following collection and stained for γH2AX to assess DNA damage response (DDR). Median fluorescence of treated samples was compared to non-irradiated control samples for each patient. Patients were characterized by observed biological kinetic response: (a) fast — phosphorylation increased by 2minutes and fell by 30minutes, (b) slow — phosphorylation continued to increase to 30minutes and (c) none — little change was observed or irradiated samples fell below controls. Total dose values were normalized to exam time for an averaged dose-rate in dose/sec for each exam. Relationships between patient biological responses and patient and exam parameters were investigated. Results: A clearer dose response at 30minutes is observed for young patients (<61yoa; R2>0.5) compared to old patients (>61yoa; R 2 <0.11). Fast responding patients were significantly younger than slow responding patients (p<0.05). Unlike in vivo samples, age did not significantly affect the patient response ex vivo. Additionally, fast responding patients received exams with significantly smaller dose-rate than slow responding patients (p<0.05). Conclusion: Age is a significant factor in the biological response suggesting that DDR may be more rapid in a younger population and slower as the population ages. Lack of an agerelated response ex vivo suggests a systemic response to radiation not present when irradiated outside the body. Dose-rate affects the biological response suggesting that patient response may be related to scan

  17. Optimal purification and sensitive quantification of DNA from fecal samples

    DEFF Research Database (Denmark)

    Jensen, Annette Nygaard; Hoorfar, Jeffrey

    2002-01-01

    Application of reliable, rapid and sensitive methods to laboratory diagnosis of zoonotic infections continues to challenge microbiological laboratories. The recovery of DNA from a swine fecal sample and a bacterial culture extracted by a conventional phenol-chloroform extraction method was compared...... = 0.99 and R-2 = 1.00). In conclusion, silica-membrane, columns can provide a more convenient and less hazardous alternative to the conventional phenol-based method. The results have implication for further improvement of sensitive amplification methods for laboratory diagnosis....

  18. Discordant genotyping results using DNA isolated from anti-doping control urine samples.

    Science.gov (United States)

    Choong, Eva; Schulze, Jenny J; Ericsson, Magnus; Rane, Anders; Ekström, Lena

    2017-07-01

    The UGT2B17 gene deletion polymorphism is known to correlate to urinary concentration of testosterone-glucuronide and hence this genotype exerts a large impact on the testosterone/epitestosterone (T/E) ratio, a biomarker for testosterone doping. The objective of this study was to assess if DNA isolated from athletes' urine samples (n = 713) obtained in routine doping controls could be targeted for genotyping analysis for future integration in the athlete's passport. A control population (n = 21) including both urine and blood DNA was used for genotyping concordance test. Another aim was to study a large group (n = 596) of authentic elite athletes in respect of urinary steroid profile in relation to genetic variation. First we found that the genotype results when using urine-derived DNA did not correlate sufficiently with the genotype obtained from whole blood DNA. Secondly we found males with one or two UGT2B17 alleles had higher T/E (mean 1.63 ± 0.93) than females (mean 1.28 ± 1.08), p˂0.001. Unexpectedly, we found that several male del/del athletes in power sports had a T/E ˃1. If men in power sport exert a different urinary steroid profile needs to be further investigated. The other polymorphisms investigated in the CYP17A1, UGT2B7 and UGT2B15 genes did not show any associations with testosterone and epitestosterone concentrations. Our results show that genotyping using urine samples according to our method is not useful in an anti-doping setting. Instead, it is of importance for the anti-doping test programs to include baseline values in the ABP to minimize any putative impact of genotype. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Development of a real-time PCR to detect Demodex canis DNA in different tissue samples.

    Science.gov (United States)

    Ravera, Ivan; Altet, Laura; Francino, Olga; Bardagí, Mar; Sánchez, Armand; Ferrer, Lluís

    2011-02-01

    The present study reports the development of a real-time polymerase chain reaction (PCR) to detect Demodex canis DNA on different tissue samples. The technique amplifies a 166 bp of D. canis chitin synthase gene (AB 080667) and it has been successfully tested on hairs extracted with their roots and on formalin-fixed paraffin embedded skin biopsies. The real-time PCR amplified on the hairs of all 14 dogs with a firm diagnosis of demodicosis and consistently failed to amplify on negative controls. Eleven of 12 skin biopsies with a morphologic diagnosis of canine demodicosis were also positive. Sampling hairs on two skin points (lateral face and interdigital skin), D. canis DNA was detected on nine of 51 healthy dogs (17.6%) a much higher percentage than previously reported with microscopic studies. Furthermore, it is foreseen that if the number of samples were increased, the percentage of positive dogs would probably also grow. Moreover, in four of the six dogs with demodicosis, the samples taken from non-lesioned skin were positive. This finding, if confirmed in further studies, suggests that demodicosis is a generalized phenomenon in canine skin, due to proliferation of local mite populations, even though macroscopic lesions only appear in certain areas. The real-time PCR technique to detect D. canis DNA described in this work is a useful tool to advance our understanding of canine demodicosis.

  20. Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients.

    Science.gov (United States)

    Millon, Laurence; Larosa, Fabrice; Lepiller, Quentin; Legrand, Faezeh; Rocchi, Steffi; Daguindau, Etienne; Scherer, Emeline; Bellanger, Anne-Pauline; Leroy, Joel; Grenouillet, Frederic

    2013-05-01

    The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.

  1. Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

    Science.gov (United States)

    Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

    2002-01-01

    Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

  2. microRNA Biomarker Discovery and High-Throughput DNA Sequencing Are Possible Using Long-term Archived Serum Samples.

    Science.gov (United States)

    Rounge, Trine B; Lauritzen, Marianne; Langseth, Hilde; Enerly, Espen; Lyle, Robert; Gislefoss, Randi E

    2015-09-01

    The impacts of long-term storage and varying preanalytical factors on the quality and quantity of DNA and miRNA from archived serum have not been fully assessed. Preanalytical and analytical variations and degradation may introduce bias in representation of DNA and miRNA and may result in loss or corruption of quantitative data. We have evaluated DNA and miRNA quantity, quality, and variability in samples stored up to 40 years using one of the oldest prospective serum collections in the world, the Janus Serumbank, a biorepository dedicated to cancer research. miRNAs are present and stable in archived serum samples frozen at -25°C for at least 40 years. Long-time storage did not reduce miRNA yields; however, varying preanalytical conditions had a significant effect and should be taken into consideration during project design. Of note, 500 μL serum yielded sufficient miRNA for qPCR and small RNA sequencing and on average 650 unique miRNAs were detected in samples from presumably healthy donors. Of note, 500 μL serum yielded sufficient DNA for whole-genome sequencing and subsequent SNP calling, giving a uniform representation of the genomes. DNA and miRNA are stable during long-term storage, making large prospectively collected serum repositories an invaluable source for miRNA and DNA biomarker discovery. Large-scale biomarker studies with long follow-up time are possible utilizing biorepositories with archived serum and state-of-the-art technology. ©2015 American Association for Cancer Research.

  3. Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

    Science.gov (United States)

    Arulandhu, Alfred J; Staats, Martijn; Hagelaar, Rico; Voorhuijzen, Marleen M; Prins, Theo W; Scholtens, Ingrid; Costessi, Adalberto; Duijsings, Danny; Rechenmann, François; Gaspar, Frédéric B; Barreto Crespo, Maria Teresa; Holst-Jensen, Arne; Birck, Matthew; Burns, Malcolm; Haynes, Edward; Hochegger, Rupert; Klingl, Alexander; Lundberg, Lisa; Natale, Chiara; Niekamp, Hauke; Perri, Elena; Barbante, Alessandra; Rosec, Jean-Philippe; Seyfarth, Ralf; Sovová, Tereza; Van Moorleghem, Christoff; van Ruth, Saskia; Peelen, Tamara; Kok, Esther

    2017-10-01

    DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance. © The Authors 2017. Published by Oxford University Press.

  4. CCR6+ Th cell distribution differentiates systemic lupus erythematosus patients based on anti-dsDNA antibody status.

    Science.gov (United States)

    Zhong, Wei; Jiang, Zhenyu; Wu, Jiang; Jiang, Yanfang; Zhao, Ling

    2018-01-01

    Systemic lupus erythematosus (SLE) disease has been shown to be associated with the generation of multiple auto-antibodies. Among these, anti-dsDNA antibodies (anti-DNAs) are specific and play a pathogenic role in SLE. Indeed, anti-DNA + SLE patients display a worse disease course. The generation of these pathogenic anti-DNAs has been attributed to the interaction between aberrant T helper (Th) cells and autoimmune B cells. Thus, in this study we have investigated whether CCR6 + Th cells have the ability to differentiate SLE patients based on anti-DNA status, and if their distribution has any correlation with disease activity. We recruited 25 anti-DNA + and 25 anti-DNA - treatment-naive onset SLE patients, matched for various clinical characteristics in our nested matched case-control study. CCR6 + Th cells and their additional subsets were analyzed in each patient by flow cytometry. Anti-DNA + SLE patients specifically had a higher percentage of Th cells expressing CCR6 and CXCR3. Further analysis of CCR6 + Th cell subsets showed that anti-DNA + SLE patients had elevated proportions of Th9, Th17, Th17.1 and CCR4/CXCR3 double-negative (DN) cells. However, the proportions of CCR6 - Th subsets, including Th1 and Th2 cells, did not show any association with anti-DNA status. Finally, we identified a correlation between CCR6 + Th subsets and clinical indicators, specifically in anti-DNA + SLE patients. Our data indicated that CCR6 + Th cells and their subsets were elevated and correlated with disease activity in anti-DNA + SLE patients. We speculated that CCR6 + Th cells may contribute to distinct disease severity in anti-DNA + SLE patients.

  5. DNA damage in blood lymphocytes in patients after {sup 177}Lu peptide receptor radionuclide therapy

    Energy Technology Data Exchange (ETDEWEB)

    Eberlein, Uta; Bluemel, Christina; Buck, Andreas Konrad; Werner, Rudolf Alexander; Lassmann, Michael [University of Wuerzburg, Department of Nuclear Medicine, Wuerzburg (Germany); Nowak, Carina; Scherthan, Harry [Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Munich (Germany)

    2015-10-15

    The aim of the study was to investigate DNA double strand break (DSB) formation and its correlation with the absorbed dose to the blood lymphocytes of patients undergoing their first peptide receptor radionuclide therapy (PRRT) with {sup 177}Lu-labelled DOTATATE/DOTATOC. The study group comprised 16 patients receiving their first PRRT. At least six peripheral blood samples were obtained before, and between 0.5 h and 48 h after radionuclide administration. From the time-activity curves of the blood and the whole body, residence times for blood self-irradiation and whole-body irradiation were determined. Peripheral blood lymphocytes were isolated, fixed with ethanol and subjected to immunofluorescence staining for colocalizing γ-H2AX/53BP1 DSB-marking foci. The average number of DSB foci per cell per patient sample was determined as a function of the absorbed dose to the blood and compared with an in vitro calibration curve established in our laboratory with {sup 131}I and {sup 177}Lu. The average number of radiation-induced foci (RIF) per cell increased over the first 5 h after radionuclide administration and decreased thereafter. A linear fit from 0 to 5 h as a function of the absorbed dose to the blood agreed with our in vitro calibration curve. At later time-points the number of RIF decreased, indicating progression of DNA repair. Measurements of RIF and the absorbed dose to the blood after systemic administration of {sup 177}Lu may be used to obtain data on the individual dose-response relationships in vivo. Individual patient data were characterized by a linear dose-dependent increase and an exponential decay function describing repair. (orig.)

  6. New Detection Modality for Label-Free Quantification of DNA in Biological Samples via Superparamagnetic Bead Aggregation

    Science.gov (United States)

    Leslie, Daniel C.; Li, Jingyi; Strachan, Briony C.; Begley, Matthew R.; Finkler, David; Bazydlo, Lindsay L.; Barker, N. Scott; Haverstick, Doris; Utz, Marcel; Landers, James P.

    2012-01-01

    Combining DNA and superparamagnetic beads in a rotating magnetic field produces multiparticle aggregates that are visually striking, and enables label-free optical detection and quantification of DNA at levels in the picogram per microliter range. DNA in biological samples can be quantified directly by simple analysis of optical images of microfluidic wells placed on a magnetic stirrer without DNA purification. Aggregation results from DNA/bead interactions driven either by the presence of a chaotrope (a nonspecific trigger for aggregation) or by hybridization with oligonucleotides on functionalized beads (sequence-specific). This paper demonstrates quantification of DNA with sensitivity comparable to that of the best currently available fluorometric assays. The robustness and sensitivity of the method enable a wide range of applications, illustrated here by counting eukaryotic cells. Using widely available and inexpensive benchtop hardware, the approach provides a highly accessible low-tech microscale alternative to more expensive DNA detection and cell counting techniques. PMID:22423674

  7. Aberrant DNA Methylation: Implications in Racial Health Disparity.

    Directory of Open Access Journals (Sweden)

    Xuefeng Wang

    Full Text Available Incidence and mortality rates of colorectal carcinoma (CRC are higher in African Americans (AAs than in Caucasian Americans (CAs. Deficient micronutrient intake due to dietary restrictions in racial/ethnic populations can alter genetic and molecular profiles leading to dysregulated methylation patterns and the inheritance of somatic to germline mutations.Total DNA and RNA samples of paired tumor and adjacent normal colon tissues were prepared from AA and CA CRC specimens. Reduced Representation Bisulfite Sequencing (RRBS and RNA sequencing were employed to evaluate total genome methylation of 5'-regulatory regions and dysregulation of gene expression, respectively. Robust analysis was conducted using a trimming-and-retrieving scheme for RRBS library mapping in conjunction with the BStool toolkit.DNA from the tumor of AA CRC patients, compared to adjacent normal tissues, contained 1,588 hypermethylated and 100 hypomethylated differentially methylated regions (DMRs. Whereas, 109 hypermethylated and 4 hypomethylated DMRs were observed in DNA from the tumor of CA CRC patients; representing a 14.6-fold and 25-fold change, respectively. Specifically; CHL1, 4 anti-inflammatory genes (i.e., NELL1, GDF1, ARHGEF4, and ITGA4, and 7 miRNAs (of which miR-9-3p and miR-124-3p have been implicated in CRC were hypermethylated in DNA samples from AA patients with CRC. From the same sample set, RNAseq analysis revealed 108 downregulated genes (including 14 ribosomal proteins and 34 upregulated genes (including POLR2B and CYP1B1 [targets of miR-124-3p] in AA patients with CRC versus CA patients.DNA methylation profile and/or products of its downstream targets could serve as biomarker(s addressing racial health disparity.

  8. Circulating Tumour DNA for Monitoring Treatment Response to Anti-PD-1 Immunotherapy in Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Atsuko Ashida

    2017-08-01

    Full Text Available Anti-programmed cell death-1 (anti-PD-1 antibody shows high therapeutic efficacy in patients with advanced melanoma. However, assessment of its therapeutic activity can be challenging because of tumour enlargement associated with intratumoural inflammation. Because circulating tumour DNA (ctDNA correlates with tumour burden, we assessed the value of ctDNA levels as an indicator of tumour changes. Quantification of ctDNA (BRAFmutant or NRASmutant levels by droplet digital PCR in 5 patients with BRAF or NRAS mutant melanoma during the treatment course showed dynamic changes corresponding to radiological and clinical alterations. In 3 cases in which the anti-PD-1 antibody was effective, ctDNA levels decreased within 2–4 weeks after treatment initiation. In 2 cases in which the anti-PD-1 antibody was ineffective, ctDNA levels did not decrease after treatment initiation. ctDNA could be a useful biomarker to predict early response to treatment in patients with advanced melanoma treated with anti-PD-1 immunotherapy.

  9. Elevated Subclinical Double-Stranded DNA Antibodies and Future Proliferative Lupus Nephritis

    Science.gov (United States)

    Lee, Jessica J.; Prince, Lisa K.; Baker, Thomas P.; Papadopoulos, Patricia; Edison, Jess; Abbott, Kevin C.

    2013-01-01

    Summary Background and objectives Elevated anti–double-stranded DNA (dsDNA) antibody and C-reactive protein are associated with proliferative lupus nephritis (PLN). Progression of quantitative anti-dsDNA antibody in patients with PLN has not been compared with that in patients with systemic lupus erythematosus (SLE) without LN before diagnosis. The temporal relationship between anti-dsDNA antibody and C-reactive protein elevation has also not been evaluated. Design, setting, participants, & measurements This case-control Department of Defense Serum Repository (established in 1985) study compared longitudinal prediagnostic quantitative anti-dsDNA antibody and C-reactive protein levels in 23 patients with biopsy-proven PLN (Walter Reed Army Medical Center, 1993–2009) with levels in 21 controls with SLE but without LN matched for patient age, sex, race, and age of serum sample. The oldest (median, 2601 days; 25%, 1245 days, 75%, 3075 days), the second to last (368; 212, 635 days), and the last (180; 135, 477 days) serum sample before diagnosis were analyzed. Results More patients with PLN had an elevated anti-dsDNA antibody level than did the matched controls at any point (78% versus 5%; P4 years (33% versus 0%; P=0.04) before diagnosis. A rate of increase >1 IU/ml per year (70% versus 0%; P<0.001) was most specific for PLN. The anti-dsDNA antibody levels increased before C-reactive protein did in most patients with an antecedent elevation (92% versus 8%; P<0.001). Conclusions Elevated anti-dsDNA antibody usually precedes both clinical and subclinical evidence of proliferative LN, which suggests direct pathogenicity. Absolute anti-dsDNA antibody level and rate of increase could better establish risk of future PLN in patients with SLE. PMID:23833315

  10. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yonghua [Iowa State Univ., Ames, IA (United States)

    2000-01-01

    Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

  11. Presence of Borrelia spp. DNA in ticks, but absence of Borrelia spp. and of Leptospira spp. DNA in blood of fever patients in Madagascar.

    Science.gov (United States)

    Hagen, Ralf Matthias; Frickmann, Hagen; Ehlers, Julian; Krüger, Andreas; Margos, Gabriele; Hizo-Teufel, Cecilia; Fingerle, Volker; Rakotozandrindrainy, Raphael; Kalckreuth, Vera von; Im, Justin; Pak, Gi Deok; Jeon, Hyon Jin; Rakotondrainiarivelo, Jean Philibert; Heriniaina, Jean Noël; Razafindrabe, Tsiry; Konings, Frank; May, Jürgen; Hogan, Benedikt; Ganzhorn, Jörg; Panzner, Ursula; Schwarz, Norbert Georg; Dekker, Denise; Marks, Florian; Poppert, Sven

    2018-01-01

    The occurrence of tick-borne relapsing fever and leptospirosis in humans in Madagascar remains unclear despite the presence of their potential vectors and reservoir hosts. We screened 255 Amblyomma variegatum ticks and 148 Rhipicephalus microplus ticks from Zebu cattle in Madagascar for Borrelia-specific DNA. Borrelia spp. DNA was detected in 21 Amblyomma variegatum ticks and 2 Rhipicephalus microplus ticks. One Borrelia found in one Rhipicephalus microplus showed close relationship to Borrelia theileri based on genetic distance and phylogenetic analyses on 16S rRNA and flaB sequences. The borreliae from Amblyomma variegatum could not be identified due to very low quantities of present DNA reflected by high cycle threshold values in real-time-PCR. It is uncertain whether these low numbers of Borrelia spp. are sufficient for transmission of infection from ticks to humans. In order to determine whether spirochaete infections are relevant in humans, blood samples of 1009 patients from the highlands of Madagascar with fever of unknown origin were screened for Borrelia spp. - and in addition for Leptospira spp. - by real-time PCR. No target DNA was detected, indicating a limited relevance of these pathogens for humans in the highlands of Madagascar. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Effects of sampling conditions on DNA-based estimates of American black bear abundance

    Science.gov (United States)

    Laufenberg, Jared S.; Van Manen, Frank T.; Clark, Joseph D.

    2013-01-01

    DNA-based capture-mark-recapture techniques are commonly used to estimate American black bear (Ursus americanus) population abundance (N). Although the technique is well established, many questions remain regarding study design. In particular, relationships among N, capture probability of heterogeneity mixtures A and B (pA and pB, respectively, or p, collectively), the proportion of each mixture (π), number of capture occasions (k), and probability of obtaining reliable estimates of N are not fully understood. We investigated these relationships using 1) an empirical dataset of DNA samples for which true N was unknown and 2) simulated datasets with known properties that represented a broader array of sampling conditions. For the empirical data analysis, we used the full closed population with heterogeneity data type in Program MARK to estimate N for a black bear population in Great Smoky Mountains National Park, Tennessee. We systematically reduced the number of those samples used in the analysis to evaluate the effect that changes in capture probabilities may have on parameter estimates. Model-averaged N for females and males were 161 (95% CI = 114–272) and 100 (95% CI = 74–167), respectively (pooled N = 261, 95% CI = 192–419), and the average weekly p was 0.09 for females and 0.12 for males. When we reduced the number of samples of the empirical data, support for heterogeneity models decreased. For the simulation analysis, we generated capture data with individual heterogeneity covering a range of sampling conditions commonly encountered in DNA-based capture-mark-recapture studies and examined the relationships between those conditions and accuracy (i.e., probability of obtaining an estimated N that is within 20% of true N), coverage (i.e., probability that 95% confidence interval includes true N), and precision (i.e., probability of obtaining a coefficient of variation ≤20%) of estimates using logistic regression. The capture probability

  13. Diagnosis of becker muscular dystrophy: Results of Re-analysis of DNA samples

    NARCIS (Netherlands)

    Straathof, Chiara S. M.; van Heusden, Dave; Ippel, Pieternella F.; Post, Jan G.; Voermans, Nicol C.; de Visser, Marianne; Brusse, Esther; van den Bergen, Janneke C.; van der Kooi, Anneke J.; Verschuuren, Jan J. G. M.; Ginjaar, Hendrika B.

    2016-01-01

    The phenotype of Becker muscular dystrophy (BMD) is highly variable, and the disease may be underdiagnosed. We searched for new mutations in the DMD gene in a cohort of previously undiagnosed patients who had been referred in the period 1985-1995. All requests for DNA analysis of the DMD gene in

  14. Clinical differences in patients with mitochondriocytopathies due to nuclear versus mitochondrial DNA mutations.

    Science.gov (United States)

    Rubio-Gozalbo, M E; Dijkman, K P; van den Heuvel, L P; Sengers, R C; Wendel, U; Smeitink, J A

    2000-01-01

    Defects in oxidative phosphorylation (OXPHOS) are genetically unique because the different components involved in this process, respiratory chain enzyme complexes (I, III, and IV) and complex V, are encoded by nuclear and mitochondrial genome. The objective of the study was to assess whether there are clinical differences in patients suffering from OXPHOS defects caused by nuclear or mitochondrial DNA (mtDNA) mutations. We studied 16 families with > or = two siblings with a genetically established OXPHOS deficiency, four due to a nuclear gene mutation and 12 due to a mtDNA mutation. Siblings with a nuclear gene mutation showed very similar clinical pictures that became manifest in the first years (ranging from first months to early childhood). There was a severe progressive course. Seven of the eight children died in their first decade. Conversely, siblings with a mtDNA mutation had clinical pictures that varied from almost alike to very distinct. They became symptomatic at an older age (ranging from childhood to adulthood), with the exception of defects associated with Leigh or Leigh-like phenotype. The clinical course was more gradual and relatively less severe; four of the 26 patients died, one in his second year, another in her second decade and two in their sixth decade. There are differences in age at onset, severity of clinical course, outcome, and intrafamilial variability in patients affected of an OXPHOS defect due to nuclear or mtDNA mutations. Patients with nuclear mutations become symptomatic at a young age, and have a severe clinical course. Patients with mtDNA mutations show a wider clinical spectrum of age at onset and severity. These differences may be of importance regarding the choice of which genome to study in affected patients as well as with respect to genetic counseling. Copyright 2000 Wiley-Liss, Inc.

  15. NMR studies of bent DNA using {sup 13}C-enriched samples

    Energy Technology Data Exchange (ETDEWEB)

    Zimmer, D.P.; Crothers, D.M. [Yale Univ., New Haven, CT (United States)

    1994-12-01

    Bending of the DNA double helix can be brought about by introducing runs of adenines (A-tracts) in phase with the helical repeat of the DNA. The requirements for bending of DNA by A-tracts are that the length of the A-tract be greater than 3 base pairs and that the A-tracts must be in phase with the helical repeat (every 10 or 11 bp). Other factors, such as the number of adenines in the run, flanking sequences, and whether the A-tracts are phased with respect to the 5{prime}A or the 3{prime}A, have effects upon the degree of bending as assayed by electrophoretic mobility on native polyacrylamide gels. There are a number of models for bending A-tract DNA. The junction-bending model postulates that the structure of A-tracts is similar to the fiber diffraction structure of poly A, in which there is a significant degree of base pair tilt with respect to the helix axis. In this model, bending occurs at the junction between the A-tract and the B-form helix to allow favorable stacking interactions to occur. The bend of the helix could arise as a result of some other perturbation of B-form DNA by A-tracts, such as propeller twist; bending also could be due to a combination of factors. Our goal is to find the structural features of A-tracts responsible for bending of the helix by performing NMR on oligonucleotides containing A-tracts to obtain higher resolution structural data. One of the problems encountered in NMR structure determination of nucleic acids and other macromolecules is the assignment of resonances to nuclei. This procedure can be greatly facilitated through the use of {sup 13}C-enriched nucleic acid samples. We are developing a technique for the enzymatic synthesis of labeled DNA for NMR. The technique we are developing is similar to RNA labeling techniques already in use. The technique involves growth of methylotrophic bacteria on {sup 13}CH{sub 3}OH.

  16. Mitochondrial DNA levels in Huntington disease leukocytes and dermal fibroblasts.

    Science.gov (United States)

    Jędrak, Paulina; Krygier, Magdalena; Tońska, Katarzyna; Drozd, Małgorzata; Kaliszewska, Magdalena; Bartnik, Ewa; Sołtan, Witold; Sitek, Emilia J; Stanisławska-Sachadyn, Anna; Limon, Janusz; Sławek, Jarosław; Węgrzyn, Grzegorz; Barańska, Sylwia

    2017-08-01

    Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.

  17. Chromatin Immunoprecipitation (ChIP): Revisiting the Efficacy of Sample Preparation, Sonication, Quantification of Sheared DNA, and Analysis via PCR

    Science.gov (United States)

    Schoppee Bortz, Pamela D.; Wamhoff, Brian R.

    2011-01-01

    The “quantitative” ChIP, a tool commonly used to study protein-DNA interactions in cells and tissue, is a difficult assay often plagued with technical error. We present, herein, the process required to merge multiple protocols into a quick, reliable and easy method and an approach to accurately quantify ChIP DNA prior to performing PCR. We demonstrate that high intensity sonication for at least 30 min is required for full cellular disruption and maximum DNA recovery because ChIP lysis buffers fail to lyse formaldehyde-fixed cells. In addition, extracting ChIP DNA with chelex-100 yields samples that are too dilute for evaluation of shearing efficiency or quantification via nanospectrophotometry. However, DNA extracted from the Mock-ChIP supernatant via the phenol-chloroform-isoamyl alcohol (PCIA) method can be used to evaluate DNA shearing efficiency and used as the standard in a fluorescence-based microplate assay. This enabled accurate quantification of DNA in chelex-extracted ChIP samples and normalization to total DNA concentration prior to performing real-time PCR (rtPCR). Thus, a quick ChIP assay that can be completed in nine bench hours over two days has been validated along with a rapid, accurate and repeatable way to quantify ChIP DNA. The resulting rtPCR data more accurately depicts treatment effects on protein-DNA interactions of interest. PMID:22046253

  18. Differences in fungi present in induced sputum samples from asthma patients and non-atopic controls: a community based case control study

    Directory of Open Access Journals (Sweden)

    van Woerden Hugo Cornelis

    2013-02-01

    Full Text Available Abstract Background There is emerging evidence for the presence of an extensive microbiota in human lungs. It is not known whether variations in the prevalence of species of microbiota in the lungs may have aetiological significance in respiratory conditions such as asthma. The aim of the study was to undertake semi-quantitative analysis of the differences in fungal species in pooled sputum samples from asthma patients and controls. Methods Induced sputum samples were collected in a case control study of asthma patients and control subjects drawn from the community in Wandsworth, London. Samples from both groups were pooled and then tested for eukaryotes. DNA was amplified using standard PCR techniques, followed by pyrosequencing and comparison of reads to databases of known sequences to determine in a semi-quantitative way the percentage of DNA from known species in each of the two pooled samples. Results A total of 136 fungal species were identified in the induced sputum samples, with 90 species more common in asthma patients and 46 species more common in control subjects. Psathyrella candolleana, Malassezia pachydermatis, Termitomyces clypeatus and Grifola sordulenta showed a higher percentage of reads in the sputum of asthma patients and Eremothecium sinecaudum, Systenostrema alba, Cladosporium cladosporioides and Vanderwaltozyma polyspora showed a higher percentage of reads in the sputum of control subjects. A statistically significant difference in the pattern of fungi that were present in the respective samples was demonstrated using the Phylogenetic (P test (P  Conclusion This study is novel in providing evidence for the widespread nature of fungi in the sputum of healthy and asthmatic individuals. Differences in the pattern of fungi present in asthma patients and controls merit further investigation. Of particular interest was the presence of Malassezia pachydermatis, which is known to be associated with atopic dermatitis.

  19. DNA methylation results depend on DNA integrity – role of post mortem interval

    Directory of Open Access Journals (Sweden)

    Mathias eRhein

    2015-05-01

    Full Text Available Major questions of neurological and psychiatric mechanisms involve the brain functions on a molecular level and cannot be easily addressed due to limitations in access to tissue samples. Post mortem studies are able to partly bridge the gap between brain tissue research retrieved from animal trials and the information derived from peripheral analysis (e.g. measurements in blood cells in patients. Here, we wanted to know how fast DNA degradation is progressing under controlled conditions in order to define thresholds for tissue quality to be used in respective trials. Our focus was on the applicability of partly degraded samples for bisulfite sequencing and the determination of simple means to define cut-off values.After opening the brain cavity, we kept two consecutive pig skulls at ambient temperature (19-21°C and removed cortex tissue up to a post mortem interval (PMI of 120h. We calculated the percentage of degradation on DNA gel electrophoresis of brain DNA to estimate quality and relate this estimation spectrum to the quality of human post-mortem control samples. Functional DNA quality was investigated by bisulfite sequencing of two functionally relevant genes for either the serotonin receptor 5 (SLC6A4 or aldehyde dehydrogenase 2 (ALDH2.Testing our approach in a heterogeneous collective of human blood and brain samples, we demonstrate integrity of measurement quality below the threshold of 72h PMI.While sequencing technically worked for all timepoints irrespective of conceivable DNA degradation, there is a good correlation between variance of methylation to degradation levels documented in the gel (R2=0.4311, p=0.0392 for advancing post mortem intervals (PMI. This otherwise elusive phenomenon is an important prerequisite for the interpretation and evaluation of samples prior to in-depth processing via an affordable and easy assay to estimate identical sample quality and thereby comparable methylation measurements.

  20. Evaluation of nested polymerase chain reaction for the early detection of Leptospira spp. DNA in serum samples from patients with leptospirosis.

    Science.gov (United States)

    Blanco, Roberta Morozetti; Romero, Eliete Caló

    2014-04-01

    The aim of this study was to analyse the nested polymerase chain reaction (nested PCR) in human serum samples of patients with clinical manifestations of leptospirosis. The cases of leptospirosis were defined by the microagglutination test (MAT). The samples were collected in 2010. Of 1042 serum samples collected from 521 patients, 28 (5.4%) were considered positive cases of leptospirosis, and 493 (94.6%) were negative. Twenty-three confirmed cases had no MAT-detectable antibodies in the acute sample (mean of 5.6 days after onset). Nested PCR was positive in 22/23 (95.7%) patients during the acute phase of the disease, with negative results by MAT. Nested PCR was negative in all convalescent serum samples with positive results by MAT. All negative cases of leptospirosis were negative by nested PCR. The nested PCR is an alternative diagnostic tool for early detection of leptospires in sera during the first 7 days of the disease. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. DNA damage in oral mucosa cells of patients with fixed orthodontic appliances.

    Directory of Open Access Journals (Sweden)

    Farzin Heravi

    2013-12-01

    Full Text Available The release of toxic metal ions from orthodontic alloys has induced concerns regarding the biocompatibility of fixed appliances. This study investigated the genotoxic effect of metal appliances in a sample of patients undergoing fixed orthodontic treatment.The study included twenty-five healthy individuals requiring orthodontic therapy in both jaws. The patients were treated by stainless steel orthodontic brackets and nickel-titanium or stainless steel arch wires. The oral mucosa cells were gathered just before the appliance placement and 9 months later. The cells were centrifuged, fixed and dropped onto slides. After staining, the micronucleus (MN assay was used to determine genome alteration. The data were analyzed by paired sample t-test.The mean micronuclei frequency in the buccal mucosa was 10.6 ± 5.7 per 1000 cells before the appliance placement and 9.2 ± 6.37 per 1000 cells 9 months later. No significant difference was found in the MN count before and 9 months after therapy (p=0.336.Under the conditions used in this study, application of fixed orthodontic appliances did not expose healthy individuals to increased risk of DNA damage in oral mucosa cells.

  2. DNA damage in leukocytes from fanconi anemia patients and heterozygotes induced by mitomycin C and ionizing radiation as assessed by the comet and comet - FISH assay

    International Nuclear Information System (INIS)

    Mohseni Meybodi, A.; Mozdarani, H.

    2009-01-01

    Lymphocytes of Fanconi anemia (FA) show an increased sensitivity to the alkylating agents such as mitomycin C (MMC), but their responses to gamma-irradiation is controversial. The extent of DNA damage in leukocytes of FA patients following irradiation and MMC treatment was studied at cellular and single chromosome level. Methods: DNA damage induced by gamma-rays and MMC was measured in leukocytes of FA patients and carriers at whole genome level using the comet assay. Also, at the DNA level of specific chromosome involved in this disease using a modified comet-FISH protocol with whole chromosome painting probes (chromosomes 16 and 13), DNA damage in leukocytes of FA patients and heterozygotes were compared to healthy individuals. Results: Baseline DNA damage in leukocytes of patients and heterozygotes was higher than in controls. Net induced DNA damage by gamma-rays in leukocytes of FA cases was not significantly different from that of healthy donors and heterozygotes. Net induced DNA damage by MMC was statistically higher and significantly different (P<0.05) in patients than other groups. Hybridization of chromosome 16 reveals more signals in the tail but the number of spots in the tail was not significantly higher than the hybridization spots for chromosome 13 in both gamma-irradiated and MMC treated samples. Conclusion: Results indicate that DNA damage induced by MMC could be a better index for diagnosis of FA patients compared to gamma-rays. Results of comet-FISH showed no difference between the sensitivity of chromosome 16 and 13 to MMC and radiation. It may indicate that, although the FA-A gene is located on chromosome 16, this chromosome might have a similar sensitivity as other chromosomes

  3. DNA damage in neurodegenerative diseases

    Energy Technology Data Exchange (ETDEWEB)

    Coppedè, Fabio, E-mail: fabio.coppede@med.unipi.it; Migliore, Lucia, E-mail: lucia.migliore@med.unipi.it

    2015-06-15

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  4. DNA damage in neurodegenerative diseases

    International Nuclear Information System (INIS)

    Coppedè, Fabio; Migliore, Lucia

    2015-01-01

    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  5. Evaluation on the effects of ageing factor, sampling and preservation methods on Asiatic black bear (Ursus thibetanus noninvasive DNA amplification

    Directory of Open Access Journals (Sweden)

    Chih-Chin SHIH

    2017-11-01

    Full Text Available Noninvasive genetic sampling allows studying wildlife without having to catch, handle or even observe individuals. In this study, factors which may affect the quality of noninvasive samples of Asiatic black bear (Ursus thibetanus in the subtropical areas were identified. We collected hair and faecal samples from captive Asiatic black bears and quantitatively evaluated the effects of hair age (from fresh to 60 days, faeces age (from fresh to 14 days, faeces sampling locations (i.e. sample collected from either the surface, inside or a mixture of both the surface and inside of faeces, and faeces preservation methods (frozen or kept at room temperature in 95% ethanol on amplification success rates of mitochondrial DNA fragments of different sizes (450bp, 900bp, and 1600bp. The results showed that the amplification success rates decreased with sample age and amplicon size in both hair and faecal DNA. In subtropical environment, there was no significant difference between amplification success of DNA extracted from fresh and 7-day-old samples of either the hair or faeces. The amplification success rates were not influenced by sampling location of faeces. For faeces preserved in 95% ethanol, the amplification success appeared unaffected by frozen at -20 °C or kept at room temperature in shorter mtDNA fragments, but was significantly influenced when amplicon size was 1600bp. The results of this study will reinforce the optimization of noninvasive sampling approaches in Asiatic black bear research, especially in the subtropics.

  6. Detection of African swine fever virus DNA in blood samples stored on FTA cards from asymptomatic pigs in Mbeya region, Tanzania.

    Science.gov (United States)

    Braae, U C; Johansen, M V; Ngowi, H A; Rasmussen, T B; Nielsen, J; Uttenthal, Å

    2015-02-01

    The aim of the study was to assess whether blood samples collected onto FTA(®) cards could be used in combination with real-time PCR for the detection of African swine fever virus (ASFV) DNA in samples from resource-poor settings under the assumption that asymptomatically (sub-clinically) infected pigs may be present. Blood samples were collected from clinically healthy pigs from Mbeya Region, Tanzania. The blood samples were stored on FTA(®) cards and analysed by real-time PCR assays in duplicate; three pigs had high levels of viral DNA (Ct values of 27-29), and three pigs had a low level of viral DNA (Ct 36-45). Four pigs were positive in one of the duplicate samples only, but clear products of the expected size were obtained when the reactions were analysed by gel electrophoresis. For comparison, blood samples from pigs experimentally infected with either a pathogenic (OURT T88/1) or a non-pathogenic (OURT T88/3) isolate of ASFV were collected, stored on FTA(®) cards and analysed in the same way. The blood from pigs infected with the OURT T88/1 isolate showed high levels of viral DNA (Ct 22-33), whereas infection with non-pathogenic OURT T88/3 isolate resulted in only low levels of viral DNA (Ct 39) in samples collected at 10-14 days after inoculation. © 2013 Blackwell Verlag GmbH.

  7. The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing.

    Science.gov (United States)

    Kennedy, Nicholas A; Walker, Alan W; Berry, Susan H; Duncan, Sylvia H; Farquarson, Freda M; Louis, Petra; Thomson, John M; Satsangi, Jack; Flint, Harry J; Parkhill, Julian; Lees, Charlie W; Hold, Georgina L

    2014-01-01

    Determining bacterial community structure in fecal samples through DNA sequencing is an important facet of intestinal health research. The impact of different commercially available DNA extraction kits upon bacterial community structures has received relatively little attention. The aim of this study was to analyze bacterial communities in volunteer and inflammatory bowel disease (IBD) patient fecal samples extracted using widely used DNA extraction kits in established gastrointestinal research laboratories. Fecal samples from two healthy volunteers (H3 and H4) and two relapsing IBD patients (I1 and I2) were investigated. DNA extraction was undertaken using MoBio Powersoil and MP Biomedicals FastDNA SPIN Kit for Soil DNA extraction kits. PCR amplification for pyrosequencing of bacterial 16S rRNA genes was performed in both laboratories on all samples. Hierarchical clustering of sequencing data was done using the Yue and Clayton similarity coefficient. DNA extracted using the FastDNA kit and the MoBio kit gave median DNA concentrations of 475 (interquartile range 228-561) and 22 (IQR 9-36) ng/µL respectively (p<0.0001). Hierarchical clustering of sequence data by Yue and Clayton coefficient revealed four clusters. Samples from individuals H3 and I2 clustered by patient; however, samples from patient I1 extracted with the MoBio kit clustered with samples from patient H4 rather than the other I1 samples. Linear modelling on relative abundance of common bacterial families revealed significant differences between kits; samples extracted with MoBio Powersoil showed significantly increased Bacteroidaceae, Ruminococcaceae and Porphyromonadaceae, and lower Enterobacteriaceae, Lachnospiraceae, Clostridiaceae, and Erysipelotrichaceae (p<0.05). This study demonstrates significant differences in DNA yield and bacterial DNA composition when comparing DNA extracted from the same fecal sample with different extraction kits. This highlights the importance of ensuring that samples

  8. Bacterial DNA in water and dialysate: detection and significance for patient outcomes.

    Science.gov (United States)

    Handelman, Garry J; Megdal, Peter A; Handelman, Samuel K

    2009-01-01

    The fluid used for hemodialysis may contain DNA fragments from bacteria, which could be harmful for patient outcomes. DNA fragments from bacteria, containing the nonmethylated CpG motif, can trigger inflammation through the monocyte and lymphocyte Toll-like receptor 9, and these DNA fragments have been observed in dialysate. The fragments may transfer across the dialyzer into the patient's bloodstream during hemodialysis treatment. During hemodiafiltration, the fragments would be introduced directly into the bloodstream. The DNA fragments may arise from biofilm in the pipes of the water system, from growth of bacteria in the water, or as contaminants in the bicarbonate and salt mixture used for preparation of dialysate. Current filtration methods, such as Diasafe filters, are not able to remove these fragments. It would be prudent to seek to reduce or eliminate these contaminants. However, the cost and effort of decreasing bacterial DNA content may ultimately require substantial facility improvements; we therefore need to fund research studies to determine if modifications to reduce bacterial DNA content are clinically warranted. This research will require methods to accurately determine the species of bacteria that contribute the DNA, since this information will allow the source to be established as biofilm, bicarbonate mixtures, or other problems in the dialysis system such as bacterial growth or leakage during water preparation. In this review, the evidence for bacterial DNA fragments will be examined and suggestions for further studies will be described.

  9. Do mtDNA Deletions Play a Role in the Development of Nasal Polyposis?

    Directory of Open Access Journals (Sweden)

    Arzu Tatar

    2014-04-01

    Full Text Available Objective:Nasal polyposis (NP is an inflammatory disease of the nasal mucosa and paranasal sinuses. Mitochondria are the cellular organelles which produce cellular energy by Oxidative Phosphorylation (OXPHOS, and they have own inheritance material, mtDNA. mtDNA is affected by reactive oxygen samples (ROS which are produced by both OXPHOS and the inflammatory process. The aim of this study was to investigate the 4977 bp and 7400 bp deletions of mtDNA in nasal polyposis tissue, and to indicate the possible association of mtDNA deletions with NP. Methods:Thirty-three patients, aged 15 to 65 years, with nasal polyposis were selected to be assessed for mitochondrial DNA deletions. The patients with possible mtDNA mutations due to mitochondrial disease, being treated with radiotherapy, of advanced age, with a familiar history, aspirin hypersensitivity, or a history of asthma, were excluded. Polyp excision surgery was applied to the treatment of the NP, and after histopathological diagnosis 1x1 cm of polyp tissue samples were used to isolate mtDNA. The 4977 bp and 7400 bp deletion regions, and two control regions of mtDNA were assessed by using four pairs of primers. DNA extractions from the NP tissues and peripheral blood samples of the patients were made, and then Polymerase Chain Reactions (PCR were made. PCR products were separated in 2% agarose gel.Results:No patient had either the 4977 bp deletion or the 7400 bp deletion in their NP tissue, and neither were these deletions evident in their peripheral blood. Two control sequences, one of them from a non-deleted region, and the other from a possible deletion region, were detected in the NP tissues and peripheral blood of all the patients.Conclusions:We had anticipated that some mtDNA deletion might have occurred in NP tissue due to the increased ROS levels caused by chronic inflammation, but we did not detect any deletion. Probably, the duration of inflammation in NP is insufficient to form mtDNA

  10. Diagnostic markers of urothelial cancer based on DNA methylation analysis

    International Nuclear Information System (INIS)

    Chihara, Yoshitomo; Hirao, Yoshihiko; Kanai, Yae; Fujimoto, Hiroyuki; Sugano, Kokichi; Kawashima, Kiyotaka; Liang, Gangning; Jones, Peter A; Fujimoto, Kiyohide; Kuniyasu, Hiroki

    2013-01-01

    Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation. In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers. Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity. Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect

  11. A Real Time PCR Platform for the Simultaneous Quantification of Total and Extrachromosomal HIV DNA Forms in Blood of HIV-1 Infected Patients

    Science.gov (United States)

    Canovari, Benedetta; Scotti, Maddalena; Acetoso, Marcello; Valentini, Massimo; Petrelli, Enzo; Magnani, Mauro

    2014-01-01

    Background The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed. Methodology/Findings Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA. Conclusions/Significance Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 104 CD4+, for 12 patients within two working days. PMID:25364909

  12. Use of SCGE method for detection of DNA comet in irradiated samples of poultry and shrimp

    International Nuclear Information System (INIS)

    Rajaei, R.; Hosseini, S.L.

    2005-01-01

    DNA in food may sustain damage by gamma irradiation.This damage can be detected by a sensitive technique, called single cell gel electrophoresis. This is a simple and low-cost technique for rapid screening of the cells of irradiated foodstuffs. For this purpose, poultry and shrimp samples were irradiated by the 60 Co gamma radiation. The radiation doses for poultry were 2,5 and 7 kGy and for shrimp were 3 and 7kGy, respectively. The irradiation samples were compared with those of unirradiated types (control). In addition, the effects of shelf-life and temperature were considered on the poultry samples only. We have found that this technique is easily applicable for identification of irradiated from unirradiated samples and it is found to be irrespective of the applied dose. It is worth mentioning that any DNA change arising from any source, for example temperature fluctuation, may be detected by the single cell gel electrophoresis technique

  13. Dysphagia is prevalent in patients with CPEO and single, large-scale deletions in mtDNA

    DEFF Research Database (Denmark)

    Pedersen, Gitte Hedermann; Løkken, Nicoline; Dahlqvist, Julia R.

    2017-01-01

    Background  The aim of this study was to assess the frequency of subjective and objective dysphagia in patients with chronic progressive external ophthalmoplegia (CPEO) due to single, large-scale deletions (LSDs) of mitochondrial DNA (mtDNA). Methods  Sixteen patients with CPEO and single LSDs...... and single LSDs of mtDNA had a prolonged cold-water test, including one with a PEG-tube, who was unable to perform the test, and nine patients reported subjective swallowing problems (56.3%). All mitochondrial myopathy patients in the control group had a normal duration of the cold-water test.  Conclusions......  The study shows that dysphagia is a common problem in patients with CPEO and LSDs of mtDNA. Dysphagia seems to be progressive with age as abnormal swallowing occurred preferentially in persons ≥ 45 years. The study shows that increased awareness of this symptom should be given to address appropriate...

  14. Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Cristina E. Stanciu

    2015-11-01

    Full Text Available The goal of this study was to investigate the relative contributions of different cellular and genetic components to biological samples created by touch or contact with a surface – one of the most challenging forms of forensic evidence. Touch samples were generated by having individuals hold an object for five minutes and analyzed for quantity of intact epidermal cells, extracellular DNA, and DNA from pelleted cell material after elution from the collection swab. Comparisons were made between samples where individuals had washed their hands immediately prior to handling and those where hand washing was not controlled. The vast majority (84-100% of DNA detected in these touch samples was extracellular and was uncorrelated to the number of epidermal cells detected. Although little to no extracellular or cell pellet-associated DNA was detected when individuals washed their hands prior to substrate handling, we found that a significant number of epidermal cells (between ~5x103 and ~1x105 could still be recovered from these samples, suggesting that other types of biological information may be present even when no amplifiable nuclear DNA is present. These results help to elucidate the biological context for touch samples and characterize factors that may contribute to patterns of transfer and persistence of genetic material in forensic evidence.

  15. Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

    Directory of Open Access Journals (Sweden)

    Praphathip Eamsobhana

    2013-02-01

    Full Text Available Cerebrospinal fluid (CSF samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10, patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5 and patients with cerebral gnathostomiasis (n = 2 and neurocysticercosis (n = 2 were examined by a single-step polymerase chain reaction (PCR method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

  16. Impaired DNA repair as assessed by the ``comet`` assay in patients developing thyroid carcinoma after radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Leprat, F.; Sarasin, A.; Suarez, H.G. [Institut de Recherches sur le Cancer, 94 - Villesuif (France); Leprat, F.; Alapetite, C.; Rosselli, F.; Ridet, A.; Moustacch, E.; Alapetite, C. [Institut Curie, 75 - Paris (France); Schlumberger, M. [Institut Gustave Roussy, 94 - Villejuif (France)

    1997-03-01

    A defective cellular response to DNA lesions induced be genotoxic agents may be associated to an increased cancer proneness. This has been clearly identified in some rare but extensively studied genetic diseases such as xeroderma pigmentosum (XP), ataxia telangiectasia (AT) and Fanconi anemia (FA). In practical oncology, most patients receive genotoxic therapeutic agents and the presence of so far unidentified sensitive genotypes could account for an increased susceptibility to cancer in a subgroup of exposed patients. The thyroid gland of children is especially sensitive to the carcinogenic effect of ionizing radiation. Evidence for risk is reported even at doses as low as 0.1 Gy, and the excess relative risk to develop a thyroid tumor following a radiation dose of 1 Gy in childhood is of 7.7 [l]. In order to determine if a defect in repair of DNA strand breaks could be involved, as an early step, in the development of secondary thyroid tumors after radiotherapy, we examined, using the alkaline single cell gel electrophoresis assay (SCGE or `comet`), the response to in vitro {gamma}-rays exposure of lymphocytes of a small group of patients who developed thyroid carcinoma after radiotherapy for a primary tumor. Because of its practical advantages, the alkaline comet assay offers the opportunity to question the role of DNA strand beaks rejoining capacity of the individual in the radiation induced carcinogenesis of thyroid tumors. This preliminary study of a small group of patients with therapeutic irradiation at childhood for a primary tumor indicates that, at the time of blood sampling, lymphocytes of some of these patients demonstrated reduced rejoining capacity. These results suggest that the comet assay might help to distinguish a subgroup of individuals at risk for radiation induced genomic instability and encourage further investigation. (authors)

  17. DNA damage in blood cells in relation to chemotherapy and nutritional status in colorectal cancer patients-A pilot study.

    Science.gov (United States)

    Kværner, Ane Sørlie; Minaguchi, Jun; Yamani, Naouale El; Henriksen, Christine; Ræder, Hanna; Paur, Ingvild; Henriksen, Hege Berg; Wiedswang, Gro; Smeland, Sigbjørn; Blomhoff, Rune; Collins, Andrew Richard; Bøhn, Siv Kjølsrud

    2018-03-01

    DNA damage can be considered as a biomarker for toxicity and response to chemotherapy. It is not known whether the chemotherapy-induced genotoxicity is associated with malnutrition. In this pilot study, we assess genotoxicity by means of DNA damage in patients with lymph-node positive colorectal cancer (CRC) and explore associations with chemotherapy treatment and nutritional status. DNA damage was compared between patients receiving chemotherapy (n = 24) and those not receiving chemotherapy (n = 20). DNA damage was measured in frozen whole blood by the comet assay. Associations between DNA damage and various indicators of malnutrition were also explored, including Patient-Generated Subjective Global Assessment (PG-SGA), bioelectrical impedance analysis (BIA) and anthropometric measurements, using multiple linear regression models. Patients on chemotherapy have higher levels of DNA damage in blood cells than patients not receiving chemotherapy (median of 16.9 and 7.9% tail DNA respectively, p = 0.001). The moderately malnourished patients (PG-SGA category B), representing 41% of the patients, have higher levels of cellular DNA damage than patients with good nutritional status (mean difference of 7.5% tail DNA, p = 0.033). In conclusion, adjuvant chemotherapy and malnutrition are both associated with increased levels of DNA damage in blood cells of CRC patients. Carefully controlled longitudinal studies or randomized controlled trials should be performed to determine whether good nutritional status may protect against chemotherapy-induced genotoxicity and enhance compliance to therapy in CRC patients. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

    Science.gov (United States)

    Diez, Cristina; Lorenz, Virginia; Ortiz, Silvia; Gonzalez, Verónica; Racca, Andrea; Bontempi, Iván; Manattini, Silvia; Solari, Aldo; Marcipar, Iván

    2010-01-01

    We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe. PMID:20064998

  19. Mitochondrial DNA (mtDNA haplogroups and serum levels of anti-oxidant enzymes in patients with osteoarthritis

    Directory of Open Access Journals (Sweden)

    Fernandez-Moreno Mercedes

    2011-11-01

    Full Text Available Abstract Background Oxidative stress play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. To prevent this, the chondrocytes possess a well-coordinated enzymatic antioxidant system. Besides, the mitochondrial DNA (mtDNA haplogroups are associated with the OA disease. Thus, the main goal of this work is to assess the incidence of the mtDNA haplogroups on serum levels of two of the main antioxidant enzymes, Manganese Superoxide Dismutase (Mn-SOD or SOD2 and catalase, and to test the suitability of these two proteins for potential OA-related biomarkers. Methods We analyzed the serum levels of SOD2 and catalase in 73 OA patients and 77 healthy controls carrying the haplogroups J, U and H, by ELISA assay. Knee and hip radiographs were classified according to Kellgren and Lawrence (K/L scoring from Grade 0 to Grade IV. Appropriate statistical analyses were performed to test the effects of clinical variables, including gender, body mass index (BMI, age, smoking status, diagnosis, haplogroups and radiologic K/L grade on serum levels of these enzymes. Results Serum levels of SOD2 appeared statistically increased in OA patients when compared with healthy controls (p Conclusions The increased levels of SOD2 in OA patients indicate an increased oxidative stress OA-related, therefore this antioxidant enzyme could be a suitable candidate biomarker for diagnosis of OA. Mitochondrial haplogroups significantly correlates with serum levels of catalase

  20. Genome-wide DNA Methylation Profiling of Cell-Free Serum DNA in Esophageal Adenocarcinoma and Barrett Esophagus

    Directory of Open Access Journals (Sweden)

    Rihong Zhai

    2012-01-01

    Full Text Available Aberrant DNA methylation (DNAm is a feature of most types of cancers. Genome-wide DNAm profiling has been performed successfully on tumor tissue DNA samples. However, the invasive procedure limits the utility of tumor tissue for epidemiological studies. While recent data indicate that cell-free circulating DNAm (cfDNAm profiles reflect DNAm status in corresponding tumor tissues, no studies have examined the association of cfDNAm with cancer or precursors on a genome-wide scale. The objective of this pilot study was to evaluate the putative significance of genome-wide cfDNAm profiles in esophageal adenocarcinoma (EA and Barrett esophagus (BE, EA precursor. We performed genome-wide DNAm profiling in EA tissue DNA (n = 8 and matched serum DNA (n = 8, in serum DNA of BE (n = 10, and in healthy controls (n = 10 using the Infinium HumanMethylation27 BeadChip that covers 27,578 CpG loci in 14,495 genes. We found that cfDNAm profiles were highly correlated to DNAm profiles in matched tumor tissue DNA (r = 0.92 in patients with EA. We selected the most differentially methylated loci to perform hierarchical clustering analysis. We found that 911 loci can discriminate perfectly between EA and control samples, 554 loci can separate EA from BE samples, and 46 loci can distinguish BE from control samples. These results suggest that genome-wide cfDNAm profiles are highly consistent with DNAm profiles detected in corresponding tumor tissues. Differential cfDNAm profiling may be a useful approach for the noninvasive screening of EA and EA premalignant lesions.

  1. Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts

    Science.gov (United States)

    Guffanti, Federica; Fratelli, Maddalena; Ganzinelli, Monica; Bolis, Marco; Ricci, Francesca; Bizzaro, Francesca; Chilà, Rosaria; Sina, Federica Paola; Fruscio, Robert; Lupia, Michela; Cavallaro, Ugo; Cappelletti, Maria Rosa; Generali, Daniele; Giavazzi, Raffaella; Damia, Giovanna

    2018-01-01

    A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients. PMID:29872499

  2. DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis

    Science.gov (United States)

    Lucena-Aguilar, Gema; Sánchez-López, Ana María; Barberán-Aceituno, Cristina; Carrillo-Ávila, José Antonio; López-Guerrero, José Antonio

    2016-01-01

    High-quality human DNA samples and associated information of individuals are necessary for biomedical research. Biobanks act as a support infrastructure for the scientific community by providing a large number of high-quality biological samples for specific downstream applications. For this purpose, biobank methods for sample preparation must ensure the usefulness and long-term functionality of the products obtained. Quality indicators are the tool to measure these parameters, the purity and integrity determination being those specifically used for DNA. This study analyzes the quality indicators in DNA samples derived from 118 frozen human tissues in optimal cutting temperature (OCT) reactive, 68 formalin-fixed paraffin-embedded (FFPE) tissues, 119 frozen blood samples, and 26 saliva samples. The results obtained for DNA quality are discussed in association with the usefulness for downstream applications and availability of the DNA source in the target study. In brief, if any material is valid, blood is the most approachable option of prospective collection of samples providing high-quality DNA. However, if diseased tissue is a requisite or samples are available, the recommended source of DNA would be frozen tissue. These conclusions will determine the best source of DNA, according to the planned downstream application. Furthermore our results support the conclusion that a complete procedure of DNA quantification and qualification is necessary to guarantee the appropriate management of the samples, avoiding low confidence results, high costs, and a waste of samples. PMID:27158753

  3. High-Risk Palliative Care Patients' Knowledge and Attitudes about Hereditary Cancer Testing and DNA Banking.

    Science.gov (United States)

    Quillin, John M; Emidio, Oluwabunmi; Ma, Brittany; Bailey, Lauryn; Smith, Thomas J; Kang, In Guk; Yu, Brandon J; Owodunni, Oluwafemi Patrick; Abusamaan, Mohammed; Razzak, Rab; Bodurtha, Joann N

    2017-12-04

    Even at the end of life, testing cancer patients for inherited susceptibility may provide life-saving information to their relatives. Prior research suggests palliative care inpatients have suboptimal understanding of genetic importance, and testing may be underutilized in this clinical setting. These conclusions are based on limited research. This study aimed to estimate genetic testing prevalence among high-risk palliative care patients in a National Cancer Institute-designated comprehensive cancer center. We also aimed to understand these patients' understanding of, and attitudes toward, hereditary cancer testing and DNA banking. Palliative care in-patients with cancer completed structured interviews, and their medical records were reviewed. Among patients at high risk for hereditary cancer, we assessed history of genetic testing/DNA banking; and related knowledge and attitudes. Among 24 high-risk patients, 14 (58.3%) said they/their relatives had genetic testing or they had been referred for a genetics consultation. Of the remaining 10 patients, seven (70%) said they would "probably" or "definitely" get tested. Patients who had not had testing were least concerned about the impact of future testing on their family relationships; two (20%) said they were "extremely concerned" about privacy related to genetic testing. Of patients without prior testing, five (50%) said they had heard or read "a fair amount" about genetic testing. No high-risk patients had banked DNA. Overall, 23 (95.8%) said they had heard or read "almost nothing" or "relatively little" about DNA banking. Written materials and clinician discussion were most preferred ways to learn about genetic testing and DNA banking. Overall, this study demonstrates underutilization of genetics services at the end of life continues to be problematic, despite high patient interest.

  4. Studies on the DNA-excision repair in lymphocytes of patients with recurrent Herpes simplex

    International Nuclear Information System (INIS)

    Fanta, D.; Topaloglou, A.; Altmann, H.

    1978-01-01

    Investigations of the semiconservatrive DNA replication and the excision repair in lymphocytes of patients with recurrent herpes simplex showed defects that could lead to mutations in the DNA with following lower immuncompetence and possibility for activation of already present oncogenic virus formations within the cellular DNA

  5. DNA extraction methods for panbacterial and panfungal PCR detection in intraocular fluids.

    Science.gov (United States)

    Mazoteras, Paloma; Bispo, Paulo José Martins; Höfling-Lima, Ana Luisa; Casaroli-Marano, Ricardo P

    2015-07-01

    Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification. Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out. The column-based extraction method was the most time-effective, achieving DNA detection limits ≥10(2) and 10(3 )CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4). The column-based extraction method for very small inocula in small volume samples (50-100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

  6. Clinical implications of monitoring circulating tumor DNA in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Schøler, Lone V; Reinert, Thomas; Ørntoft, Mai-Britt W

    2017-01-01

    .007). Changes in ctDNA levels induced by relapse intervention (n = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman ρ = 0.4).Conclusions: Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance...

  7. Detection of Babesia DNA in blood and spleen samples from Eurasian badgers (Meles meles) in Scotland.

    Science.gov (United States)

    Bartley, Paul M; Wilson, Cari; Innes, Elisabeth A; Katzer, Frank

    2017-08-01

    Babesia are intraerythrocytic parasites of importance worldwide within the fields of human and veterinary medicine, as some Babesia sp., including Babesia microti are potentially zoonotic and can cause fatal disease in both humans and animals. The aims of this study were to use a nested PCR (amplifying the 18S rRNA gene) to determine the presence and species of Babesia parasite DNA found in blood (n = 47) and spleen (n = 47) samples collected from Eurasian badgers (Meles meles) in Scotland. The results showed 28/47 (59·6%) blood and 14/47 (29·8%) spleen samples tested positive for the presence of Babesia DNA. Initial sequence analysis of the Babesia DNA identified three distinct sequence types (submitted to GenBank KX528553, KX528554 and KX528555), which demonstrated ⩾99% identity to Babesia sp. parasites previously identified in badgers in Spain (KT223484 and KT223485). Phylogenetic analysis showed that the three isolates are closely related to Babesia annae, B. microti and other Piroplasmida species found in wildlife. Further sequence analysis of the samples demonstrated that the badgers were routinely infected with more than one parasite isolate and there was also evidence of genetic recombination between the Babesia parasite isolates (submitted to GenBank KY250472 - KY250477).

  8. Preferential recognition of auto-antibodies against 4-hydroxynonenal modified DNA in the cancer patients.

    Science.gov (United States)

    Faisal, Mohammad; Shahab, Uzma; Alatar, Abdulrahman A; Ahmad, Saheem

    2017-11-01

    The structural perturbations in DNA molecule may be caused by a break in a strand, a missing base from the backbone, or a chemically changed base. These alterations in DNA that occurs naturally can result from metabolic or hydrolytic processes. DNA damage plays a major role in the mutagenesis, carcinogenesis, aging and various other patho-physiological conditions. DNA damage can be induced through hydrolysis, exposure to reactive oxygen species (ROS) and other reactive carbonyl metabolites including 4-hydroxynonenal (HNE). 4-HNE is an important lipid peroxidation product which has been implicated in the mutagenesis and carcinogenesis processes. The present study examines to probe the presence of auto-antibodies against 4-hydroxynonenal damaged DNA (HNE-DNA) in various cancer subjects. In this study, the purified calf thymus DNA was damaged by the action of 4-HNE. The DNA was incubated with 4-HNE for 24 h at 37°C temperature. The binding characteristics of cancer auto-antibodies were assessed by direct binding and competitive inhibition ELISA. DNA modifications produced hyperchromicity in UV spectrum and decreased fluorescence intensity. Cancer sera exhibited enhanced binding with the 4-HNE modified calf thymus DNA as compared to its native conformer. The 4-HNE modified DNA presents unique epitopes which may be one of the factors for the auto-antibody induction in cancer patients. The HNE modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients. © 2017 Wiley Periodicals, Inc.

  9. Circulating bacterial-derived DNA fragment level is a strong predictor of cardiovascular disease in peritoneal dialysis patients.

    Directory of Open Access Journals (Sweden)

    Cheuk-Chun Szeto

    Full Text Available Circulating bacterial DNA fragment is related to systemic inflammatory state in peritoneal dialysis (PD patients. We hypothesize that plasma bacterial DNA level predicts cardiovascular events in new PD patients.We measured plasma bacterial DNA level in 191 new PD patients, who were then followed for at least a year for the development of cardiovascular event, hospitalization, and patient survival.The average age was 59.3 ± 11.8 years; plasma bacterial DNA level 34.9 ± 1.5 cycles; average follow up 23.2 ± 9.7 months. At 24 months, the event-free survival was 86.1%, 69.8%, 55.4% and 30.8% for plasma bacterial DNA level quartiles I, II, III and IV, respectively (p < 0.0001. After adjusting for confounders, plasma bacterial DNA level, baseline residual renal function and malnutrition-inflammation score were independent predictors of composite cardiovascular end-point; each doubling in plasma bacterial DNA level confers a 26.9% (95% confidence interval, 13.0 - 42.5% excess in risk. Plasma bacterial DNA also correlated with the number of hospital admission (r = -0.379, p < 0.0001 and duration of hospitalization for cardiovascular reasons (r = -0.386, p < 0.0001. Plasma bacterial DNA level did not correlate with baseline arterial pulse wave velocity (PWV, but with the change in carotid-radial PWV in one year (r = -0.238, p = 0.005.Circulating bacterial DNA fragment level is a strong predictor of cardiovascular event, need of hospitalization, as well as the progressive change in arterial stiffness in new PD patients.

  10. Automation and integration of multiplexed on-line sample preparation with capillary electrophoresis for DNA sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Tan, H.

    1999-03-31

    The purpose of this research is to develop a multiplexed sample processing system in conjunction with multiplexed capillary electrophoresis for high-throughput DNA sequencing. The concept from DNA template to called bases was first demonstrated with a manually operated single capillary system. Later, an automated microfluidic system with 8 channels based on the same principle was successfully constructed. The instrument automatically processes 8 templates through reaction, purification, denaturation, pre-concentration, injection, separation and detection in a parallel fashion. A multiplexed freeze/thaw switching principle and a distribution network were implemented to manage flow direction and sample transportation. Dye-labeled terminator cycle-sequencing reactions are performed in an 8-capillary array in a hot air thermal cycler. Subsequently, the sequencing ladders are directly loaded into a corresponding size-exclusion chromatographic column operated at {approximately} 60 C for purification. On-line denaturation and stacking injection for capillary electrophoresis is simultaneously accomplished at a cross assembly set at {approximately} 70 C. Not only the separation capillary array but also the reaction capillary array and purification columns can be regenerated after every run. DNA sequencing data from this system allow base calling up to 460 bases with accuracy of 98%.

  11. Studies on the DNA excision repair in lymphocytes of patients with recurrent herpes simplex

    International Nuclear Information System (INIS)

    Fanta, D.; Topaloglou, A.; Altmann, H.

    1979-01-01

    DNA repair was investigated in lymphocytes from patients with recurrent herpes simplex and from healthy controls. From the results - depressed UV type repair, depressed gamma type repair, reduced RF - it may be concluded that mutations can be expected due to the faults remaining in the DNA. This may not only lower cellular immunocompetence, but also activate already present oncogenic virus informations within the cellular DNA. Thus, irrespective of the possible oncogenic potential of HSV, there seems to be an increased risk of late effects in patients with recurrent herpetic manifestations. (Auth.)

  12. The use of DNA fingerprinting to resolve conflicting results in patients with suspected gastrointestinal malignancy.

    Science.gov (United States)

    Islam, Sameer; Miller, Ethan D; Patel, Neal; De Petris, Giovanni; Highsmith, Edward W; Fleischer, David E

    2013-03-01

    To underscore the utility of DNA fingerprinting for clarifying disparate results from endoscopic pathologic specimens. Occasionally, serially obtained gastrointestinal biopsies may yield inconsistent results. These discrepancies pose a dilemma for gastroenterologists and their patients, especially when malignancy is a consideration. Patients referred to our tertiary care center from outside institutions had undergone endoscopically obtained esophageal biopsies showing malignancy, verified by pathologists at both our site and from the referring center. Repeat endoscopic biopsies at our center did not show malignancy. To verify that different sets of biopsies came from the same patient, we performed a polymerase chain reaction-based analysis comparing the 2 specimens. This analysis, called DNA fingerprinting, can show a high degree of certainty whether 2 specimens came from the same patient. In each case, DNA fingerprinting verified a match, laying the groundwork for intervention. One patient underwent endoscopic radiofrequency ablation to the esophageal mucosa involved. Another underwent esophagectomy with partial gastrectomy. Both are doing well clinically and remain cancer-free on follow-up. DNA fingerprinting is a powerful and a relatively inexpensive tool. Usually, only small amounts of tissue are required, and even degraded or archival tissue is adequate. DNA fingerprinting can be an important tool in the gastroenterologist's arsenal to help clarify conflicting results, allowing the patient and physician to move forward with the management.

  13. Study of DNA damage with a new system for irradiation of samples in a nuclear reactor

    Energy Technology Data Exchange (ETDEWEB)

    Gual, Maritza R., E-mail: mrgual@instec.c [Instituto Superior de Tecnologias y Ciencias Aplicadas, InSTEC, Avenida Salvador Allende y Luaces, Quinta de Los Molinos, Plaza de la Revolucion, Havana, AP 6163 (Cuba); Milian, Felix M. [Universidade Estadual de Santa Cruz, UESC (Brazil); Deppman, Airton [Instituto de Fisica, Universidad de Sao Paulo, IF-USP, Rua do Matao, Travessa R, no. 187, Ciudade Universitaria, Butanta, CEP 05508-900, Sao Paulo (Brazil); Coelho, Paulo R.P. [Instituto de Pesquisas Energeticas e Nucleares, IPEN-CNEN/SP (Brazil)

    2011-02-15

    In this paper, we report results of a quantitative analysis of the effects of neutrons on DNA, and, specifically, the production of simple and double breaks of plasmid DNA in aqueous solutions with different concentrations of free-radical scavengers. The radiation damage to DNA was evaluated by electrophoresis through agarose gels. The neutron and gamma doses were measured separately with thermoluminescent detectors. In this work, we have also demonstrated usefulness of a new system for positioning and removing samples in channel BH3 of the IEA-R1 reactor at the Instituto de Pesquisas Energeticas e Nucleares (Brazil) without necessity of interrupting the reactor operation.

  14. DNA-damage foci to detect and characterize DNA repair alterations in children treated for pediatric malignancies.

    Directory of Open Access Journals (Sweden)

    Nadine Schuler

    Full Text Available PURPOSE: In children diagnosed with cancer, we evaluated the DNA damage foci approach to identify patients with double-strand break (DSB repair deficiencies, who may overreact to DNA-damaging radio- and chemotherapy. In one patient with Fanconi anemia (FA suffering relapsing squamous cell carcinomas of the oral cavity we also characterized the repair defect in biopsies of skin, mucosa and tumor. METHODS AND MATERIALS: In children with histologically confirmed tumors or leukemias and healthy control-children DSB repair was investigated by counting γH2AX-, 53BP1- and pATM-foci in blood lymphocytes at defined time points after ex-vivo irradiation. This DSB repair capacity was correlated with treatment-related normal-tissue responses. For the FA patient the defective repair was also characterized in tissue biopsies by analyzing DNA damage response proteins by light and electron microscopy. RESULTS: Between tumor-children and healthy control-children we observed significant differences in mean DSB repair capacity, suggesting that childhood cancer is based on genetic alterations affecting DNA repair. Only 1 out of 4 patients with grade-4 normal-tissue toxicities revealed an impaired DSB repair capacity. The defective DNA repair in FA patient was verified in irradiated blood lymphocytes as well as in non-irradiated mucosa and skin biopsies leading to an excessive accumulation of heterochromatin-associated DSBs in rapidly cycling cells. CONCLUSIONS: Analyzing human tissues we show that DSB repair alterations predispose to cancer formation at younger ages and affect the susceptibility to normal-tissue toxicities. DNA damage foci analysis of blood and tissue samples allows one to detect and characterize DSB repair deficiencies and enables identification of patients at risk for high-grade toxicities. However, not all treatment-associated normal-tissue toxicities can be explained by DSB repair deficiencies.

  15. Gamma-H2Ax quantification of low dose irradiation-induced DNA damage in patients receiving intensity modulated radiotherapy (IRMT)

    International Nuclear Information System (INIS)

    Sivabalasingham, S.; Short, S.; Worku, M.; Marks, G.; Guerrero-Urbano, T.

    2013-01-01

    The full text of the publication follows. Purpose/Objective: IMRT (Intensity Modulated Radiotherapy) offers greater target dose compliance yet may produce a comparative higher whole body dose. The aim of this study is to quantify γH2Ax foci in lymphocytes (an established marker of DNA double strand breaks) in patients undergoing IMRT. Material/Methods: Radical inverse planned IMRT was delivered to patients with brain tumours. Peripheral blood samples were collected from each patient at the following time points: baseline; weekly- prior to and 30 minutes after one treatment fraction; 2 and 6 weeks following completion of treatment. Whole blood was centrifuged to separate lymphocytes, which were fixed and stained for fluorescent immunocytochemistry. 150 cells per sample were visualized. γH2Ax foci were identified and counted using confocal microscopy. Results A low basal level of foci was present in all samples prior to any radiation exposure (0.233, SD 0.028). There was a significant increase in mean foci per cell in post radiotherapy treatment samples(0.367 foci per cell pre-treatment and 0.612 foci per cell post treatment, p=0.000) and no significant difference between post-treatment foci numbers at different times during treatment(for example, 0.518 foci per cell at week 1 and 0.760 at week 6, p=0.279). Mean foci numbers returned to background levels at 6 weeks following completion of radiotherapy (0.239 foci per cell at baseline and 0.219 foci per cell at 6 weeks, p=0.529). Comparison between patients treated with different delivery methods is ongoing. Conclusion: γH2Ax is a feasible marker of DNA damage in lymphocytes during IMRT. These data demonstrate a reproducible level of foci induction in patients undergoing IMRT for tumour targets in brain. There is no significant accumulation of foci during treatment and foci numbers return to baseline post treatment. This assay may be useful to assess differences in whole body dose when different delivery methods

  16. Prevalence of JC virus in Chinese patients with colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Xiaozhou Mou

    Full Text Available BACKGROUND: JCV is a DNA polyomavirus very well adapted to humans. Although JCV DNA has been detected in colorectal cancers (CRC, the association between JCV and CRC remains controversial. In China, the presence of JCV infection in CRC patients has not been reported. Here, we investigated JCV infection and viral DNA load in Chinese CRC patients and to determine whether the JCV DNA in peripheral blood (PB can be used as a diagnostic marker for JCV-related CRC. METHODOLOGY/PRINCIPAL FINDINGS: Tumor tissues, non-cancerous tumor-adjacent tissues and PB samples were collected from 137 CRC patients. In addition, 80 normal colorectal tissue samples from patients without CRC and PB samples from 100 healthy volunteers were also harvested as controls. JCV DNA was detected by nested PCR and glass slide-based dot blotting. Viral DNA load of positive samples were determined by quantitative real-time PCR. JCV DNA was detected in 40.9% (56/137 of CRC tissues at a viral load of 49.1 to 10.3×10(4 copies/µg DNA. Thirty-four (24.5% non-cancerous colorectal tissues (192.9 to 4.4×10(3 copies/µg DNA and 25 (18.2% PB samples (81.3 to 4.9×10(3 copies/µg DNA from CRC patients were positive for JCV. Tumor tissues had higher levels of JCV than non-cancerous tissues (P = 0.003 or PB samples (P<0.001. No correlation between the presence of JCV and demographic or medical characteristics was observed. The JCV prevalence in PB samples was significantly associated with the JCV status in tissue samples (P<0.001. Eleven (13.8% normal colorectal tissues and seven (7.0% PB samples from healthy donors were positive for JCV. CONCLUSIONS/SIGNIFICANCE: JCV infection is frequently present in colorectal tumor tissues of CRC patients. Although the association between JCV presence in PB samples and JCV status in tissue samples was identified in this study, whether PB JCV detection can serve as a marker for JCV status of CRC requires further study.

  17. Decreased UV-induced DNA repair synthesis in peripheral leukocytes from patients with the nevoid basal cell carcinoma syndrome

    International Nuclear Information System (INIS)

    Ringborg, U.; Lambert, B.; Landergen, J.; Lewensohn, R.

    1981-01-01

    The uv-induced DNA repair synthesis in peripheral leukocytes from 7 patients with the nevoid basal cell carcinoma syndrome was compared to that in peripheral leukocytes from 5 patients with basal cell carcinomas and 39 healthy subjects. A dose response curve was established for each individual, and maximum DNA repair synthesis was used as a measure of the capacity for DNA repair. The patients with the nevoid basal cell carcinoma syndrome had about 25% lower level of maximum DNA repair synthesis as compared to the patients with basal cell carcinomas and control individuals. The possibility that DNA repair mechanisms may be involved in the etiology to the nevoid basal cell carcinoma syndrome is discussed

  18. Value of urinary topoisomerase-IIA cell-free DNA for diagnosis of bladder cancer.

    Science.gov (United States)

    Kim, Ye-Hwan; Yan, Chunri; Lee, Il-Seok; Piao, Xuan-Mei; Byun, Young Joon; Jeong, Pildu; Kim, Won Tae; Yun, Seok-Joong; Kim, Wun-Jae

    2016-03-01

    Topoisomerase-II alpha (TopoIIA ), a DNA gyrase isoform that plays an important role in the cell cycle, is present in normal tissues and various human cancers, and can show altered expression in both. The aim of the current study was to examine the value of urinary TopoIIA cell-free DNA as a noninvasive diagnosis of bladder cancer (BC). Two patient cohorts were examined. Cohort 1 (73 BC patients and seven controls) provided bladder tissue samples, whereas cohort 2 (83 BC patients, 54 nonmalignant hematuric patients, and 61 normal controls) provided urine samples. Real-time quantitative polymerase chain reaction was used to measure expression of TopoIIA mRNA in tissues and TopoIIA cell-free DNA in urine samples. The results showed that expression of TopoIIA mRNA in BC tissues was significantly higher than that in noncancer control tissues (pbladder cancer (MIBC) when compared with nonmuscle invasive bladder cancer (NMIBC) (p=0.002). Receiver operating characteristics (ROC) curve analysis was performed to examine the sensitivity/specificity of urinary TopoIIA cell-free DNA for diagnosing BC, NMIBC, and MIBC. The areas under the ROC curve for BC, NMIBC, and MIBC were 0.741, 0.701, and 0.838, respectively. In summary, the results of this study provide evidence that cell-free TopoIIA DNA may be a potential biomarker for BC.

  19. Presence of herpesvirus DNA in cerebrospinal fluid of patients with tick-borne encephalitis and enteroviral meningoencephalitis.

    Science.gov (United States)

    Labská, Klára; Roubalová, Kateřina; Pícha, Dušan; Marešová, Vilma

    2015-07-01

    Reactivation of HHVs in the CNS due to inflammation has not been well described yet. The primary aim of this study was to investigate the frequency of HHV DNA detection in the cerebrospinal fluid (CSF) of immunocompetent patients with meningoencephalitis of other than HHV origin. The secondary aim of this study was to evaluate the impact of herpesvirus co-infection on the clinical course and patient outcome. Ninety-six patients with clinically and laboratory proven tick-borne encephalitis (TBE) and 77 patients with a confirmed diagnosis of enteroviral meningitis (EVM), along with a control group of 107 patients without evidence of inflammation in the CSF were retrospectively tested by nested PCR for the presence of DNA of the neurotropic herpesviruses HSV1, HSV2, VZV, and HHV6 in the CSF. The clinical course, laboratory tests, antiviral treatment, and neurological complications in a 6-month follow-up were compared between the groups positive or negative for HHV DNA in the CSF. HHV DNA was found in the CSF of 12 (6.9%) patients (6.3% and 7.8% in the TBE and EVM groups, respectively) and in 1 (0.9%) control patient. None of the patients had recent blisters or rash. The clinical course was comparably mild in all patients. No permanent neurological sequelae were observed. Only the CSF total protein level was significantly higher in HHV DNA-positive than in HHV-negative patients. © 2015 Wiley Periodicals, Inc.

  20. Acetone facilitated DNA sampling from electrical tapes improves DNA recovery and enables latent fingerprints development.

    Science.gov (United States)

    Feine, Ilan; Shpitzen, Moshe; Geller, Boris; Salmon, Eran; Peleg, Tsach; Roth, Jonathan; Gafny, Ron

    2017-07-01

    Electrical tapes (ETs) are a common component of improvised explosive devices (IEDs) used by terrorists or criminal organizations and represent a valuable forensic resource for DNA and latent fingerprints recovery. However, DNA recovery rates are typically low and usually below the minimal amount required for amplification. In addition, most DNA extraction methods are destructive and do not allow further latent fingerprints development. In the present study a cell culture based touch DNA model was used to demonstrate a two-step acetone-water DNA recovery protocol from ETs. This protocol involves only the adhesive side of the ET and increases DNA recovery rates by up to 70%. In addition, we demonstrated partially successful latent fingerprints development from the non-sticky side of the ETs. Taken together, this protocol maximizes the forensic examination of ETs and is recommended for routine casework processing. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. DNA barcoding of selected UAE medicinal plant species: a comparative assessment of herbarium and fresh samples.

    Science.gov (United States)

    Enan, Mohamed Rizk; Palakkott, Abdul Rasheed; Ksiksi, Taoufik Saleh

    2017-01-01

    It is commonly difficult to extract and amplify DNA from herbarium samples as they are old and preserved using different compounds. In addition, such samples are subjected to the accumulation of intrinsically produced plant substances over long periods (up to hundreds of years). DNA extraction from desert flora may pause added difficulties as many contain high levels of secondary metabolites. Herbarium samples from the Biology Department (UAE University) plant collection and fresh plant samples, collected from around Al-Ain (UAE), were used in this study. The three barcode loci for the coding genes matK, rbcL and rpoC1-were amplified. Our results showed that T. terresteris , H. robustum , T. pentandrus and Z. qatarense were amplified using all three primers for both fresh and herbaium samples. Both fresh and herbarium samples of C. comosum , however, were not amplified at all, using the three primers. Herbarium samples from A. javanica , C. imbricatum , T. aucherana and Z. simplex were not amplified with any of the three primers. For fresh samples 90, 90 and 80% of the samples were amplified using matK, rbcL and rpoC1, respectively. In short, fresh samples were significantly better amplified than those from herbarium sources, using the three primers. Both fresh and herbarium samples from one species ( C. comosum ), however, were not successfully amplified. It is also concluded that the rbcL regions showed real potentials to distinguish the UAE species under investigation into the appropriate family and genus.

  2. Mitochondrial DNA triplication and punctual mutations in patients with mitochondrial neuromuscular disorders

    Energy Technology Data Exchange (ETDEWEB)

    Mkaouar-Rebai, Emna, E-mail: emna.mkaouar@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Felhi, Rahma; Tabebi, Mouna [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Alila-Fersi, Olfa; Chamkha, Imen [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Maalej, Marwa; Ammar, Marwa [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Kammoun, Fatma [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Keskes, Leila [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Hachicha, Mongia [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Fakhfakh, Faiza, E-mail: faiza.fakhfakh02@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia)

    2016-04-29

    Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes of complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder. - Highlights: • We reported 2 patients with mitochondrial neuromuscular disorders. • The heteroplasmic MT-ATP6 9157G>A variation was reported. • A triplication of 9 bp in the mitochondrial NC7 region was detected. • The m.14924T>C transition (S60P) in the MT-CYB gene was found.

  3. Methylated DNA for monitoring tumor growth and regression

    DEFF Research Database (Denmark)

    Kristiansen, Søren; Nielsen, Dorte; Söletormos, Georg

    2014-01-01

    Abstract A wide range of protein cancer biomarkers is currently recommended in international guidelines for monitoring the growth and regression of solid tumors. However, a number of these markers are also present in low concentrations in blood obtained from healthy individuals and from patients...... of gene promoters. Because tumor cells naturally secrete DNA and upon cell death leak DNA, modified methylated DNA can be detected in blood, urine, sputum and other body fluids. At present international guidelines do not include recommendations for monitoring modified methylated DNA. The low level...... of evidence can partly be explained by incomplete collection of serial blood samples, by analytical challenges, and by lack of knowledge of how monitoring studies should be designed and how serial marker data obtained from individual patients should be interpreted. Here, we review the clinical validity...

  4. Human papillomavirus DNA and p16 expression in Japanese patients with oropharyngeal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Kawakami, Hisato; Okamoto, Isamu; Terao, Kyoichi; Sakai, Kazuko; Suzuki, Minoru; Ueda, Shinya; Tanaka, Kaoru; Kuwata, Kiyoko; Morita, Yume; Ono, Koji; Nishio, Kazuto; Nishimura, Yasumasa; Doi, Katsumi; Nakagawa, Kazuhiko

    2013-01-01

    Human papillomavirus (HPV) is a major etiologic factor for oropharyngeal squamous cell carcinoma (OPSCC). However, little is known about HPV-related OPSCC in Japan. During the study, formalin-fixed, paraffin-embedded OPSCC specimens from Japanese patients were analyzed for HPV DNA by the polymerase chain reaction (PCR) and for the surrogate marker p16 by immuno-histochemistry. For HPV DNA-positive, p16-negative specimens, the methylation status of the p16 gene promoter was examined by methylation-specific PCR. Overall survival was calculated in relation to HPV DNA and p16 status and was subjected to multivariate analysis. OPSCC cell lines were examined for sensitivity to radiation or cisplatin in vitro. The study results showed that tumor specimens from 40 (38%) of the 104 study patients contained HPV DNA, with such positivity being associated with tumors of the tonsils, lymph node metastasis, and nonsmoking. Overall survival was better for OPSCC patients with HPV DNA than for those without it (hazard ratio, 0.214; 95% confidence interval, 0.074–0.614; P = 0.002). Multivariate analysis revealed HPV DNA to be an independent prognostic factor for overall survival (P = 0.015). Expression of p16 was associated with HPV DNA positivity. However, 20% of HPV DNA-positive tumors were negative for p16, with most of these tumors manifesting DNA methylation at the p16 gene promoter. Radiation or cisplatin sensitivity did not differ between OPSCC cell lines positive or negative for HPV DNA. Thus, positivity for HPV DNA identifies a distinct clinical subset of OPSCC with a more favorable outcome in Japanese

  5. A differential autophagy-dependent response to DNA double-strand breaks in bone marrow mesenchymal stem cells from sporadic ALS patients

    Directory of Open Access Journals (Sweden)

    Shane Wald-Altman

    2017-05-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is an incurable motor neurodegenerative disease caused by a diversity of genetic and environmental factors that leads to neuromuscular degeneration and has pathophysiological implications in non-neural systems. Our previous work showed abnormal levels of mRNA expression for biomarker genes in non-neuronal cell samples from ALS patients. The same genes proved to be differentially expressed in the brain, spinal cord and muscle of the SOD1G93A ALS mouse model. These observations support the idea that there is a pathophysiological relevance for the ALS biomarkers discovered in human mesenchymal stem cells (hMSCs isolated from bone marrow samples of ALS patients (ALS-hMSCs. Here, we demonstrate that ALS-hMSCs are also a useful patient-based model to study intrinsic cell molecular mechanisms of the disease. We investigated the ALS-hMSC response to oxidative DNA damage exerted by neocarzinostatin (NCS-induced DNA double-strand breaks (DSBs. We found that the ALS-hMSCs responded to this stress differently from cells taken from healthy controls (HC-hMSCs. Interestingly, we found that ALS-hMSC death in response to induction of DSBs was dependent on autophagy, which was initialized by an increase of phosphorylated (pAMPK, and blocked by the class III phosphoinositide 3-kinase (PI3K and autophagy inhibitor 3-methyladenine (3MeA. ALS-hMSC death in response to DSBs was not apoptotic as it was caspase independent. This unique ALS-hMSC-specific response to DNA damage emphasizes the possibility that an intrinsic abnormal regulatory mechanism controlling autophagy initiation exists in ALS-patient-derived hMSCs. This mechanism may also be relevant to the most-affected tissues in ALS. Hence, our approach might open avenues for new personalized therapies for ALS.

  6. A differential autophagy-dependent response to DNA double-strand breaks in bone marrow mesenchymal stem cells from sporadic ALS patients.

    Science.gov (United States)

    Wald-Altman, Shane; Pichinuk, Edward; Kakhlon, Or; Weil, Miguel

    2017-05-01

    Amyotrophic lateral sclerosis (ALS) is an incurable motor neurodegenerative disease caused by a diversity of genetic and environmental factors that leads to neuromuscular degeneration and has pathophysiological implications in non-neural systems. Our previous work showed abnormal levels of mRNA expression for biomarker genes in non-neuronal cell samples from ALS patients. The same genes proved to be differentially expressed in the brain, spinal cord and muscle of the SOD1 G93A ALS mouse model. These observations support the idea that there is a pathophysiological relevance for the ALS biomarkers discovered in human mesenchymal stem cells (hMSCs) isolated from bone marrow samples of ALS patients (ALS-hMSCs). Here, we demonstrate that ALS-hMSCs are also a useful patient-based model to study intrinsic cell molecular mechanisms of the disease. We investigated the ALS-hMSC response to oxidative DNA damage exerted by neocarzinostatin (NCS)-induced DNA double-strand breaks (DSBs). We found that the ALS-hMSCs responded to this stress differently from cells taken from healthy controls (HC-hMSCs). Interestingly, we found that ALS-hMSC death in response to induction of DSBs was dependent on autophagy, which was initialized by an increase of phosphorylated (p)AMPK, and blocked by the class III phosphoinositide 3-kinase (PI3K) and autophagy inhibitor 3-methyladenine (3MeA). ALS-hMSC death in response to DSBs was not apoptotic as it was caspase independent. This unique ALS-hMSC-specific response to DNA damage emphasizes the possibility that an intrinsic abnormal regulatory mechanism controlling autophagy initiation exists in ALS-patient-derived hMSCs. This mechanism may also be relevant to the most-affected tissues in ALS. Hence, our approach might open avenues for new personalized therapies for ALS. © 2017. Published by The Company of Biologists Ltd.

  7. Tracing Males From Different Continents by Genotyping JC Polyomavirus in DNA From Semen Samples.

    Science.gov (United States)

    Rotondo, John Charles; Candian, Tommaso; Selvatici, Rita; Mazzoni, Elisa; Bonaccorsi, Gloria; Greco, Pantaleo; Tognon, Mauro; Martini, Fernanda

    2017-05-01

    The human JC polyomavirus (JCPyV) is an ubiquitous viral agent infecting approximately 60% of humans. Recently, JCPyV sequences have been detected in semen samples. The aim of this investigation was to test whether semen JCPyV genotyping can be employed to trace the origin continent of males. Semen DNA samples (n = 170) from males of different Continents were investigated by PCR for the polymorphic JCPyV viral capsid protein 1 (VP1) sequences, followed by DNA sequencing. JCPyV sequences were detected with an overall prevalence of 27.6% (47/170). DNA sequencing revealed that European males carried JCPyV types 1A (71.4%), 4 (11.4%), 2B (2.9%), 2D1 (2.9%), and 3A (2.9%). Asians JCPyV type 2D1 (66.7%) and Africans JCPyV types 3A (33.3%) and 1A (33.3%). In 10.6% of males, two different JCPyV genotypes were detected, suggesting that the second JCPyV genotype was acquired in the destination country. This study indicates that the majority of semen samples found to be JCPyV-positive, were infected with the JCPyV genotype found in the geographic area of male origin. Therefore, semen JCPyV genotyping could be employed to trace the origin continent of males. Our findings could be applied to forensic investigations, in case of for instance sexual crimes. Indeed, JCPyV genotyping should enable investigators to make additional detailed profiling of the offender. J. Cell. Physiol. 232: 982-985, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    2012-01-01

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study, DNA was extracted either directly or following freeze storage of three homogenized human fecal...

  9. Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Bergström, Anders; Licht, Tine Rask

    Freezing stool samples prior to DNA extraction and downstream analysis is widely used in metagenomic studies of the human microbiota but may affect the inferred community composition. In this study DNA was extracted either directly or following freeze storage of three homogenized human fecal...

  10. Exploitation of FTA cartridges for the sampling, long-term storage, and DNA-based analyses of plant-parasitic nematodes.

    Science.gov (United States)

    Marek, Martin; Zouhar, Miloslav; Douda, Ondřej; Maňasová, Marie; Ryšánek, Pavel

    2014-03-01

    The use of DNA-based analyses in molecular plant nematology research has dramatically increased over recent decades. Therefore, the development and adaptation of simple, robust, and cost-effective DNA purification procedures are required to address these contemporary challenges. The solid-phase-based approach developed by Flinders Technology Associates (FTA) has been shown to be a powerful technology for the preparation of DNA from different biological materials, including blood, saliva, plant tissues, and various human and plant microbial pathogens. In this work, we demonstrate, for the first time, that this FTA-based technology is a valuable, low-cost, and time-saving approach for the sampling, long-term archiving, and molecular analysis of plant-parasitic nematodes. Despite the complex structure and anatomical organization of the multicellular bodies of nematodes, we report the successful and reliable DNA-based analysis of nematode high-copy and low-copy genes using the FTA technology. This was achieved by applying nematodes to the FTA cards either in the form of a suspension of individuals, as intact or pestle-crushed nematodes, or by the direct mechanical printing of nematode-infested plant tissues. We further demonstrate that the FTA method is also suitable for the so-called "one-nematode-assay", in which the target DNA is typically analyzed from a single individual nematode. More surprisingly, a time-course experiment showed that nematode DNA can be detected specifically in the FTA-captured samples many years after initial sampling occurs. Collectively, our data clearly demonstrate the applicability and the robustness of this FTA-based approach for molecular research and diagnostics concerning phytonematodes; this research includes economically important species such as the stem nematode (Ditylenchus dipsaci), the sugar beet nematode (Heterodera schachtii), and the Northern root-knot nematode (Meloidogyne hapla).

  11. DNA barcoding of authentic and substitute samples of herb of the family Asparagaceae and Asclepiadaceae based on the ITS2 region

    Directory of Open Access Journals (Sweden)

    Padmalatha S Rai

    2012-01-01

    Full Text Available Background : Herbal drugs used to treat illness according to Ayurveda are often misidentified or adulterated with similar plant materials. Objective: To aid taxonomical identification, we used DNA barcoding to evaluate authentic and substitute samples of herb and phylogenetic relationship of four medicinal plants of family Asparagaceace and Asclepiadaceae. Materials and Methods : DNA extracted from dry root samples of two authentic and two substitutes of four specimens belonging to four species were subjected to polymerase chain reaction (PCR and DNA sequencing. Primers for nuclear DNA (nu ITS2 and plastid DNA (matK and rpoC1 were used for PCR and sequence analysis was performed by Clustal W. The intraspecific variation and interspecific divergence were calculated using MEGA V 4.0. Statistical Analysis : Kimura′s two parameter model, neighbor joining and bootstrapping methods were used in this work. Results: The result indicates the efficiency of amplification for ITS2 candidate DNA barcodes was 100% for four species tested. The average interspecific divergence is 0.12 and intraspecific variation was 0.232 in the case of two Asparagaceae species. In two Asclepiadaceae species, average interspecific divergence and intraspecific variation were 0.178 and 0.004 respectively. Conclusions: Our findings show that the ITS2 region can effectively discriminate Asparagus racemosus and Hemidesmus indicus from its substitute samples and hence can resolve species admixtures in raw samples. The ITS2 region may be used as one of the standard DNA barcodes to identify closely related species of family Asclepiadaceae but was noninformative for Asparagaceae species suggesting a need for the development of new markers for each family. More detailed studies involving more species and substitutes are warranted.

  12. Evaluation of DNA damage in patients with arsenic poisoning: urinary 8-hydroxydeoxyguanine

    International Nuclear Information System (INIS)

    Yamauchi, Hiroshi; Aminaka, Yoshito; Yoshida, Katsumi; Sun Guifan; Pi Jingbo; Waalkes, Michael P.

    2004-01-01

    The relationship between arsenic exposure and DNA damage in patients with acute or chronic arsenic poisoning was analyzed. Urinary 8-hydroxydeoxyguanine (8-OHdG) concentrations were measured as an indication of oxidative DNA damage. A remarkable increase in 8-OHdG in the urine was observed in 60% of 52 patients with acute arsenic poisoning from the accidental oral intake of the arsenic trioxide. This was two- to threefold higher than levels in normal healthy subjects (n = 248). There was a clear relationship between arsenic concentrations in urine after acute poisoning and elevated levels of 8-OHdG. Levels of urinary 8-OHdG returned to normal within 180 days after the acute arsenic poisoning event. In patients chronically poisoned by the consumption of well water with elevated levels of arsenate [As(V)], elevated 8-OHdG concentrations in urine were also observed. A significant correlation between the 8-OHdG levels and arsenic levels in the urine was observed in 82 patients with chronic poisoning. Thus, evidence of oxidative DNA damage occurred in acute arsenic poisoning by arsenite [As(III)] and in chronic arsenic poisoning by As(V). In chronic poisoning patients provided low-arsenic drinking water, evidence of DNA damage subsided between 9 months and 1 year after the high levels of arsenic intake were reduced. The initial level of arsenic exposure appeared to dictate the length of this recovery period. These data indicate that some aspects of chronic and acute arsenic poisoning may be reversible with the cessation of exposure. This knowledge may contribute to our understanding of the risk elevation from arsenic carcinogenesis and perhaps be used in a prospective fashion to assess individual risk

  13. DNA stable-isotope probing (DNA-SIP).

    Science.gov (United States)

    Dunford, Eric A; Neufeld, Josh D

    2010-08-02

    DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

  14. Evaluation of HPV DNA positivity in colorectal cancer patients in Kerman, Southeast Iran

    Science.gov (United States)

    Malekpour Afshar, Reza; Deldar, Zeinab; Mollaei, Hamid Reza; Arabzadeh, Seyed Alimohammad; Iranpour, Maryam

    2018-01-27

    Background: The HPV virus is known to be oncogenic and associations with many cancers has been proven. Although many studies have been conducted on the possible relationship with colorectal cancer (CRC), a definitive role of the virus has yet to be identified. Method: In this cross-sectional study, the frequency of HPV positivity in CRC samples in Kerman was assessed in 84 cases with a mean age of 47.7 ± 12.5 years over two years. Qualitative real time PCR was performed using general primers for the L1 region of HPV DNA. Results: Out of 84 CRC samples, 19 (22.6%), proved positive for HPV DNA. Genotyping of positive samples showed all of these to be of high risk HPV type. Prevalence of HPV infection appears to depend geographic region, life style, diet and other factors. Conclusion: In our location frequency of CRC is low, and this limited the sample size for evaluation of HPV DNA. The most prevalent types were HPV types 51 and 56. While HPV infection may play an important role in colorectal carcinogenesis, this needs to be assessed in future studies. Creative Commons Attribution License

  15. Next-generation sequencing-based detection of circulating tumour DNA After allogeneic stem cell transplantation for lymphoma.

    Science.gov (United States)

    Herrera, Alex F; Kim, Haesook T; Kong, Katherine A; Faham, Malek; Sun, Heather; Sohani, Aliyah R; Alyea, Edwin P; Carlton, Victoria E; Chen, Yi-Bin; Cutler, Corey S; Ho, Vincent T; Koreth, John; Kotwaliwale, Chitra; Nikiforow, Sarah; Ritz, Jerome; Rodig, Scott J; Soiffer, Robert J; Antin, Joseph H; Armand, Philippe

    2016-12-01

    Next-generation sequencing (NGS)-based circulating tumour DNA (ctDNA) detection is a promising monitoring tool for lymphoid malignancies. We evaluated whether the presence of ctDNA was associated with outcome after allogeneic haematopoietic stem cell transplantation (HSCT) in lymphoma patients. We studied 88 patients drawn from a phase 3 clinical trial of reduced-intensity conditioning HSCT in lymphoma. Conventional restaging and collection of peripheral blood samples occurred at pre-specified time points before and after HSCT and were assayed for ctDNA by sequencing of the immunoglobulin or T-cell receptor genes. Tumour clonotypes were identified in 87% of patients with adequate tumour samples. Sixteen of 19 (84%) patients with disease progression after HSCT had detectable ctDNA prior to progression at a median of 3·7 months prior to relapse/progression. Patients with detectable ctDNA 3 months after HSCT had inferior progression-free survival (PFS) (2-year PFS 58% vs. 84% in ctDNA-negative patients, P = 0·033). In multivariate models, detectable ctDNA was associated with increased risk of progression/death (Hazard ratio 3·9, P = 0·003) and increased risk of relapse/progression (Hazard ratio 10·8, P = 0·0006). Detectable ctDNA is associated with an increased risk of relapse/progression, but further validation studies are necessary to confirm these findings and determine the clinical utility of NGS-based minimal residual disease monitoring in lymphoma patients after HSCT. © 2016 John Wiley & Sons Ltd.

  16. Extraction of Total DNA and RNA from Marine Filter Samples and Generation of a cDNA as Universal Template for Marker Gene Studies.

    Science.gov (United States)

    Schneider, Dominik; Wemheuer, Franziska; Pfeiffer, Birgit; Wemheuer, Bernd

    2017-01-01

    Microbial communities play an important role in marine ecosystem processes. Although the number of studies targeting marker genes such as the 16S rRNA gene has been increased in the last few years, the vast majority of marine diversity is rather unexplored. Moreover, most studies focused on the entire bacterial community and thus disregarded active microbial community players. Here, we describe a detailed protocol for the simultaneous extraction of DNA and RNA from marine water samples and for the generation of cDNA from the isolated RNA which can be used as a universal template in various marker gene studies.

  17. DNA profiling of trace DNA recovered from bedding.

    Science.gov (United States)

    Petricevic, Susan F; Bright, Jo-Anne; Cockerton, Sarah L

    2006-05-25

    Trace DNA is often detected on handled items and worn clothing examined in forensic laboratories. In this study, the potential transfer of trace DNA to bedding by normal contact, when an individual sleeps in a bed, is examined. Volunteers slept one night on a new, lower bed sheet in their own bed and one night in a bed foreign to them. Samples from the sheets were collected and analysed by DNA profiling. The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples. This transfer may have important repercussions in sexual assault investigations.

  18. Histopathological examination of nerve samples from pure neural leprosy patients: obtaining maximum information to improve diagnostic efficiency.

    Science.gov (United States)

    Antunes, Sérgio Luiz Gomes; Chimelli, Leila; Jardim, Márcia Rodrigues; Vital, Robson Teixeira; Nery, José Augusto da Costa; Corte-Real, Suzana; Hacker, Mariana Andréa Vilas Boas; Sarno, Euzenir Nunes

    2012-03-01

    Nerve biopsy examination is an important auxiliary procedure for diagnosing pure neural leprosy (PNL). When acid-fast bacilli (AFB) are not detected in the nerve sample, the value of other nonspecific histological alterations should be considered along with pertinent clinical, electroneuromyographical and laboratory data (the detection of Mycobacterium leprae DNA with polymerase chain reaction and the detection of serum anti-phenolic glycolipid 1 antibodies) to support a possible or probable PNL diagnosis. Three hundred forty nerve samples [144 from PNL patients and 196 from patients with non-leprosy peripheral neuropathies (NLN)] were examined. Both AFB-negative and AFB-positive PNL samples had more frequent histopathological alterations (epithelioid granulomas, mononuclear infiltrates, fibrosis, perineurial and subperineurial oedema and decreased numbers of myelinated fibres) than the NLN group. Multivariate analysis revealed that independently, mononuclear infiltrate and perineurial fibrosis were more common in the PNL group and were able to correctly classify AFB-negative PNL samples. These results indicate that even in the absence of AFB, these histopathological nerve alterations may justify a PNL diagnosis when observed in conjunction with pertinent clinical, epidemiological and laboratory data.

  19. Blood global DNA methylation is decreased in non-severe chronic obstructive pulmonary disease (COPD) patients.

    Science.gov (United States)

    Zinellu, Angelo; Sotgiu, Elisabetta; Fois, Alessandro G; Zinellu, Elisabetta; Sotgia, Salvatore; Ena, Sara; Mangoni, Arduino A; Carru, Ciriaco; Pirina, Pietro

    2017-10-01

    Alterations in global DNA methylation have been associated with oxidative stress (OS). Since chronic obstructive pulmonary disease (COPD) is characterized by increased oxidative stress we aimed to evaluate the levels of global DNA methylation in this patient group. We assessed methylcytosine (mCyt) levels in DNA from blood collected in 43 COPD patients (29 with mild and 14 with moderate disease) and 43 age- and sex-matched healthy controls. DNA methylation was significantly lower in COPD patients vs. controls (4.20 ± 0.18% mCyt vs. 4.29 ± 0.18% mCyt, p = 0.02). Furthermore, DNA methylation in COPD patients with moderate disease was significantly lower than that in patients with mild disease (4.14 ± 0.15% mCyt vs. 4.23 ± 0.19% mCyt, p COPD (crude OR = 0.06, 95% CI 0.00 to 0.67, p = 0.023). This relationship remained significant after adjusting for several confounders (OR 0.03, 95% CI 0.00 to 0.67; p = 0.028). Receiver operating characteristics (ROC) curve analysis demonstrated the area under the curve of mCyt was 0.646, with 46.6% sensitivity and 79.1% specificity for presence of COPD. There were no significant correlations between methylation and OS indices. The presence and severity of COPD is associated with progressively lower DNA methylation in blood. However, this epigenetic alteration seems independent of oxidative stress. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Comparison of Two Molecular Assays for Detection and Characterization of Aspergillus fumigatus Triazole Resistance and Cyp51A Mutations in Clinical Isolates and Primary Clinical Samples of Immunocompromised Patients

    Directory of Open Access Journals (Sweden)

    Patricia Postina

    2018-03-01

    Full Text Available In hematological patients, the incidence of invasive aspergillosis (IA caused by azole resistant Aspergillus fumigatus (ARAf is rising. As the diagnosis of IA is rarely based on positive culture in this group of patients, molecular detection of resistance mutations directly from clinical samples is crucial. In addition to the in-house azole resistance ARAf polymerase chain reaction (PCR assays detecting the frequent mutation combinations TR34/L98H, TR46/Y121F/T289A, and M220 in the Aspergillus fumigatus (A. fumigatus Cyp51A gene by subsequent DNA sequence analysis, we investigated in parallel the commercially available AsperGenius® real time PCR system in detecting the Cyp51A alterations TR34/L98H and Y121F/T289A directly from 52 clinical samples (15 biopsies, 22 bronchoalveolar lavage (BAL, 15 cerebrospinal fluid (CSF samples and ARAf isolates (n = 3 of immunocompromised patients. We analyzed DNA aliquots and compared both methods concerning amplification and detection of Aspergillus DNA and Cyp51A alterations. As positive control for the feasibility of our novel Y121F and T289A PCR assays, we used two A. fumigatus isolates with the TR46/Y121F/T289A mutation combination isolated from hematological patients with known Cyp51A alterations and a lung biopsy sample of a patient with acute myeloid leukemia (AML. The rate of positive ARAf PCR results plus successful sequencing using the ARAf PCR assays was 61% in biopsies, 29% in CSF, 67% in BAL samples and 100% in isolates. In comparison the amount of positive PCRs using the AsperGenius® assays was 47% in biopsies, 42% in CSF, 59% in BAL samples and 100% in isolates. Altogether 17 Cyp51A alterations were detected using our ARAf PCRs plus DNA sequencing and therefrom 10 alterations also by the AsperGenius® system. The comparative evaluation of our data revealed that our conventional PCR assays are more sensitive in detecting ARAf in BAL and biopsy samples, whereby differences were not significant

  1. Absence of Mycoplasma-specific DNA sequence in brain, blood and CSF of patients with multiple sclerosis (MS): a study by PCR and real-time PCR.

    Science.gov (United States)

    Casserly, Georgina; Barry, Thomas; Tourtellotte, Wallace W; Hogan, Edward L

    2007-02-15

    Mycoplasmas are the smallest of the known self-replicating organisms. They lack cell walls and are associated with numerous diseases in humans and animals. We are exploring the possibility that infection by Mycoplasma may induce the inflammatory demyelinating disease of the central nervous system (CNS) that is MS. The presence of specific Mycoplasma species DNA was sought in brain, serum and cerebrospinal fluid (CSF) of patients diagnosed with multiple sclerosis (MS) and other neurological diseases (OND) including inflammatory disorders. The MS samples from patients with active and progressive MS, as well as in remission, a variety of other neurological disease controls, including inflammatory CNS diseases such as meningitis, cryptococcal meningitis and encephalitis and other neurological disorders such as migraine were also examined. Clinical samples were provided by the National Neurological Research Specimen Bank and the Human Brain and Spinal Fluid Resource Centre, Los Angeles. Analysis was carried out by conventional PCR using Mycoplasma-specific primers (McAuliffe et al., 2005) that target the 16S rDNA gene in Mycoplasma species. The Mycoplasma-specific primers could detect 102 Mycoplasma species. In this study, 30 samples of human brain and 57 pairs of serum and CSF and were examined. No Mycoplasma-specific nucleic acid sequence was detected, and the consistent observation of an endogenous gene, human serum albumin (HSA), as a positive control documented the adequacy of the method. Real-time PCR analysis of serum and CSF was done also targeting utilizing the Mycoplasma 16S rDNA gene, and this also demonstrated the lack of Mycoplasma in these samples. The presence of Mycoplasma at extraneural sites in MS patients is now being explored.

  2. Analysis of twelve polymorphous bookmarks in the DNA of a population sample of the Costa Rican Central Valley

    International Nuclear Information System (INIS)

    Rojas, E.; Lobo, J.; Leon, P.

    1999-01-01

    To establish databases of allele frequencies in a Costa Rican Central Valley population sample. Peripheral blood samples from more than 40 individual were used to isolate DNA and analyze each sample with 10 dinucleotide repeat genetic markers and with 2 mini satellite repeats, using the polymerase chain reaction. Alleles were identified by comparison with DNA from CEPH family members. Genotypes were determined by labelling one of the two Pcr primers with 32P before amplification, electrophoresis in sequencing gels and autoradiography. Analysis of this data set indicates that these samples is in Hardy-Weinberg equilibrium and shows no evidence of linkage disequilibrium between markers. These data are compared with results from other human populations analyzed with the same markers, finding similarities in allele frequencies among them. Notably, the Costa Rican sample presents the lowest heterozygosity value, with 4 of the 10 dinucleotide markers tested, followed by a Cerdenian sample. In contrast, the two African samples presented the highest heterozygosity indexes with a larger number of alleles. (L. Jimenez) [es

  3. Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

    Science.gov (United States)

    Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

    2018-06-13

    The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

  4. eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

    Science.gov (United States)

    Liu, Robin H.; Longiaru, Mathew

    2009-05-01

    DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

  5. Sequence analysis of the canine mitochondrial DNA control region from shed hair samples in criminal investigations.

    Science.gov (United States)

    Berger, C; Berger, B; Parson, W

    2012-01-01

    In recent years, evidence from domestic dogs has increasingly been analyzed by forensic DNA testing. Especially, canine hairs have proved most suitable and practical due to the high rate of hair transfer occurring between dogs and humans. Starting with the description of a contamination-free sample handling procedure, we give a detailed workflow for sequencing hypervariable segments (HVS) of the mtDNA control region from canine evidence. After the hair material is lysed and the DNA extracted by Phenol/Chloroform, the amplification and sequencing strategy comprises the HVS I and II of the canine control region and is optimized for DNA of medium-to-low quality and quantity. The sequencing procedure is based on the Sanger Big-dye deoxy-terminator method and the separation of the sequencing reaction products is performed on a conventional multicolor fluorescence detection capillary electrophoresis platform. Finally, software-aided base calling and sequence interpretation are addressed exemplarily.

  6. Quantification of circulating cell-free DNA in the serum of patients with obstructive sleep apnea-hypopnea syndrome.

    Science.gov (United States)

    Ye, Liang; Ma, Guan-Hua; Chen, Ling; Li, Min; Liu, Jia-Lin; Yang, Kun; Li, Qing-Yun; Li, Ning; Wan, Huan-Ying

    2010-12-01

    Serum cell-free DNA concentrations have been reported to increase in many acute diseases as well as in some chronic conditions such as cancer and autoimmune diseases. The aim of this study was to examine whether serum DNA concentrations were elevated in patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). The effects of nasal continuous positive airway pressure (nCPAP) on serum DNA were also investigated. One hundred twenty-seven people diagnosed with OSAHS by polysomnography (PSG) were admitted into the OSAHS group, and 52 subjects without OSAHS were recruited for the control group. The OSAHS group was further divided into mild, moderate, and severe OSAHS subgroups based on their apnea-hypopnea index (AHI) during sleep. Ten patients with moderate and severe OSAHS were treated with nCPAP. Serum DNA, interleukin-6 (IL-6), and malonaldehyde (MDA) concentrations were measured and were found to be significantly higher in patients with moderate and severe OSAHS groups than those in the mild OSAHS and control groups (p DNA correlated positively with AHI, oxygen desaturation index (ODI), IL-6, and MDA, and negatively correlated with minimal oxygen saturation (miniSaO(2)) (all p DNA concentrations. After 6 months of nCPAP therapy, serum concentrations of DNA, IL-6, and MDA were significantly decreased (p DNA in patients with OSAHS was positively correlated with disease severity. Serum DNA may become an important parameter for monitoring the severity of OSAHS and effectiveness of therapy.

  7. Small sample whole-genome amplification

    Science.gov (United States)

    Hara, Christine; Nguyen, Christine; Wheeler, Elizabeth; Sorensen, Karen; Arroyo, Erin; Vrankovich, Greg; Christian, Allen

    2005-11-01

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  8. Identification of the Mislabeled Breast Cancer Samples by Mitochondrial DNA Haplotyping

    Directory of Open Access Journals (Sweden)

    Xiaogang Chen

    2015-01-01

    Full Text Available The task to identify whether an archival malignant tumor specimen had been mislabeled or interchanged is a challenging one for forensic genetics. The nuclear DNA (nDNA markers were affected by the aberration of tumor cells, so they were not suitable for personal identification when the tumor tissues were tested. In this study, we focused on a new solution - mitochondrial single nucleotide polymorphism (mtSNP haplotyping by a multiplex SNaPshot assay. To validate our strategy of haplotyping with 25 mtSNPs, we analyzed 15 pairs of cancerous/healthy tissues taken from patients with ductal breast carcinoma. The haplotypes of all the fifteen breast cancer tissues were matched with their paired breast tissues. The heteroplasmy at 2 sites, 14783A/G and 16519C/T was observed in one breast tissue, which indicated a mixture of related mitochondrial haplotypes. However, only one haplotype was retained in the paired breast cancer tissue, which could be considered the result of proliferation of tumor subclone. The allele drop-out and allele drop-in were observed when 39 STRs and 20 tri-allelic SNPs of nDNA were applied. Compared to nDNA markers applied, 25 mtSNPs were more stable without interference from aberrance of breast cancer. Also, two cases were presented where the investigation of haplotype with 25 mtSNPs was used to prove the origin of biopsy specimen with breast cancer. The mislabeling of biopsy specimen with breast cancer could be certified in one case but could not be supported in the other case. We highlight the importance of stability of mtSNP haplotype in breast cancer. It was implied that our multiplex SNaPshot assay with 25 mtSNPs was a useful strategy to identify mislabeled breast cancer specimen.

  9. Differences in Bordetella pertussis DNA load according to clinical and epidemiological characteristics of patients with whooping cough.

    Science.gov (United States)

    Brotons, Pedro; de Paz, Hector D; Toledo, Diana; Villanova, Marta; Plans, Pedro; Jordan, Iolanda; Dominguez, Angela; Jane, Mireia; Godoy, Pere; Muñoz-Almagro, Carmen

    2016-04-01

    To identify associations between nasopharyngeal Bordetella pertussis DNA load and clinical and epidemiological characteristics and evaluate DNA load prognostic value in pertussis severity. Prospective observational multi-centre study including nasopharyngeal samples positive to pertussis DNA by real-time PCR collected from children and adult patients in more than 200 health centres of Catalonia (Spain) during 2012-2013. B. pertussis load was inversely correlated with age (rho = -0.32, p < 0.001), time to diagnosis (rho = -0.33, p < 0.001) and number of symptoms (rho = 0.13, p = 0.002). Median bacterial load was significantly higher in inpatients versus outpatients (4.91 vs. 2.55 log10 CFU/mL, p < 0.001), patients with complications versus those without (6.05 vs. 2.82 log10 CFU/mL, p < 0.001), disease incidence in summer and autumn versus spring and winter (3.50 vs. 2.21 log10 CFU/mL, p = 0.002), and unvaccinated-partially vaccinated patients versus vaccinated (4.20 vs. 2.76 log10 CFU/mL, p = 0.004). A logistic regression model including bacterial load and other candidate prognostic factors showed good prediction for hospital care (AUC = 0.94) although only age and unvaccinated status were found to be prognostic factors. We observed strong positive associations of nasopharyngeal bacterial load with severity outcomes of hospitalisation and occurrence of complications. Bacterial load and other independent variables contributed to an accurate prognostic model for hospitalisation. Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  10. Mendelian breeding units versus standard sampling strategies: mitochondrial DNA variation in southwest Sardinia

    Directory of Open Access Journals (Sweden)

    Daria Sanna

    2011-01-01

    Full Text Available We report a sampling strategy based on Mendelian Breeding Units (MBUs, representing an interbreeding group of individuals sharing a common gene pool. The identification of MBUs is crucial for case-control experimental design in association studies. The aim of this work was to evaluate the possible existence of bias in terms of genetic variability and haplogroup frequencies in the MBU sample, due to severe sample selection. In order to reach this goal, the MBU sampling strategy was compared to a standard selection of individuals according to their surname and place of birth. We analysed mitochondrial DNA variation (first hypervariable segment and coding region in unrelated healthy subjects from two different areas of Sardinia: the area around the town of Cabras and the western Campidano area. No statistically significant differences were observed when the two sampling methods were compared, indicating that the stringent sample selection needed to establish a MBU does not alter original genetic variability and haplogroup distribution. Therefore, the MBU sampling strategy can be considered a useful tool in association studies of complex traits.

  11. Genotyping of samples from German patients with ocular, cerebral and systemic toxoplasmosis reveals a predominance of Toxoplasma gondii type II.

    Science.gov (United States)

    Herrmann, Daland C; Maksimov, Pavlo; Hotop, Andrea; Groß, Uwe; Däubener, Walter; Liesenfeld, Oliver; Pleyer, Uwe; Conraths, Franz J; Schares, Gereon

    2014-10-01

    Toxoplasmosis is an important zoonosis transmitted from animals to humans world-wide. In order to determine Toxoplasma gondii genotypes in individuals living in Germany and to compare findings with those in animals, we analysed nine independent and unlinked genetic markers (nSAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) by PCR-RFLP in 83 archived T. gondii-positive DNA samples from patients with ocular toxoplasmosis (n=35), toxoplasmic encephalitis (n=32), systemic toxoplasmosis after bone-marrow transplantation (n=15) and congenital toxoplasmosis (n=1). In 46 of these 83 samples the presence of T. gondii DNA was confirmed by conventional end-point PCR. Among these, 17 T. gondii-positive samples were typed at all nine loci. The majority (15/17, 88.2%) of these samples were of T. gondii type II (i.e., including both, the Apico type II and Apico type I variants). In addition, in one sample a T. gondii type II/type III allele combination and in another sample a T. gondii genotype displaying type III alleles at all markers was observed. In the remaining 11 samples, in which T. gondii could only be partially typed, exclusively type II (n=10) or type III (n=1) alleles were observed. Results of the present study suggest that the majority of patients in Germany are infected with type II T. gondii regardless of the clinical manifestation of toxoplasmosis. This finding is in accord with the predominance of type II T. gondii in oocysts isolated from cats and in tissues of other intermediate hosts in Germany. Copyright © 2014 Elsevier GmbH. All rights reserved.

  12. Viral-Cellular DNA Junctions as Molecular Markers for Assessing Intra-Tumor Heterogeneity in Cervical Cancer and for the Detection of Circulating Tumor DNA

    Directory of Open Access Journals (Sweden)

    Katrin Carow

    2017-09-01

    Full Text Available The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.

  13. Back to basics: an evaluation of NaOH and alternative rapid DNA extraction protocols for DNA barcoding, genotyping, and disease diagnostics from fungal and oomycete samples.

    Science.gov (United States)

    Osmundson, Todd W; Eyre, Catherine A; Hayden, Katherine M; Dhillon, Jaskirn; Garbelotto, Matteo M

    2013-01-01

    The ubiquity, high diversity and often-cryptic manifestations of fungi and oomycetes frequently necessitate molecular tools for detecting and identifying them in the environment. In applications including DNA barcoding, pathogen detection from plant samples, and genotyping for population genetics and epidemiology, rapid and dependable DNA extraction methods scalable from one to hundreds of samples are desirable. We evaluated several rapid extraction methods (NaOH, Rapid one-step extraction (ROSE), Chelex 100, proteinase K) for their ability to obtain DNA of quantity and quality suitable for the following applications: PCR amplification of the multicopy barcoding locus ITS1/5.8S/ITS2 from various fungal cultures and sporocarps; single-copy microsatellite amplification from cultures of the phytopathogenic oomycete Phytophthora ramorum; probe-based P. ramorum detection from leaves. Several methods were effective for most of the applications, with NaOH extraction favored in terms of success rate, cost, speed and simplicity. Frozen dilutions of ROSE and NaOH extracts maintained PCR viability for over 32 months. DNA from rapid extractions performed poorly compared to CTAB/phenol-chloroform extracts for TaqMan diagnostics from tanoak leaves, suggesting that incomplete removal of PCR inhibitors is an issue for sensitive diagnostic procedures, especially from plants with recalcitrant leaf chemistry. NaOH extracts exhibited lower yield and size than CTAB/phenol-chloroform extracts; however, NaOH extraction facilitated obtaining clean sequence data from sporocarps contaminated by other fungi, perhaps due to dilution resulting from low DNA yield. We conclude that conventional extractions are often unnecessary for routine DNA sequencing or genotyping of fungi and oomycetes, and recommend simpler strategies where source materials and intended applications warrant such use. © 2012 Blackwell Publishing Ltd.

  14. Challenging genosensors in food samples: The case of gluten determination in highly processed samples.

    Science.gov (United States)

    Martín-Fernández, Begoña; de-los-Santos-Álvarez, Noemí; Martín-Clemente, Juan Pedro; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

    2016-01-01

    Electrochemical genosensors have undergone an enormous development in the last decades, but only very few have achieved a quantification of target content in highly processed food samples. The detection of allergens, and particularly gluten, is challenging because legislation establishes a threshold of 20 ppm for labeling as gluten-free but most genosensors expresses the results in DNA concentration or DNA copies. This paper describes the first attempt to correlate the genosensor response and the wheat content in real samples, even in the case of highly processed food samples. A sandwich-based format, comprising a capture probe immobilized onto the screen-printed gold electrode, and a signaling probe functionalized with fluorescein isothiocyanate (FITC), both hybridizing with the target was used. The hybridization event was electrochemically monitored by adding an anti-FITC peroxidase (antiFITC-HRP) and its substrate, tetramethylbenzidine. Binary model mixtures, as a reference material, and real samples have been analyzed. DNA from food was extracted and a fragment encoding the immunodominant peptide of α2-gliadin amplified by a tailored PCR. The sensor was able to selectively detect toxic cereals for celiac patients, such as different varieties of wheat, barley, rye and oats, from non-toxic plants. As low as 0.001% (10 mg/kg) of wheat flour in an inert matrix was reliably detected, which directly compete with the current method of choice for DNA detection, the real-time PCR. A good correlation with the official immunoassay was found in highly processed food samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Direct quantification of cell-free, circulating DNA from unpurified plasma.

    Science.gov (United States)

    Breitbach, Sarah; Tug, Suzan; Helmig, Susanne; Zahn, Daniela; Kubiak, Thomas; Michal, Matthias; Gori, Tommaso; Ehlert, Tobias; Beiter, Thomas; Simon, Perikles

    2014-01-01

    Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.

  16. DNA preservation in silk.

    Science.gov (United States)

    Liu, Yawen; Zheng, Zhaozhu; Gong, He; Liu, Meng; Guo, Shaozhe; Li, Gang; Wang, Xiaoqin; Kaplan, David L

    2017-06-27

    The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50-70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

  17. Post-cardiac arrest level of free-plasma DNA and DNA-histone complexes

    DEFF Research Database (Denmark)

    Jeppesen, A N; Hvas, A-M; Grejs, A M

    2017-01-01

    Background Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone...... after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISAplus. The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals. Results We found no difference...... in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P

  18. The influence of radio- and chemotherapy on DNA repair of peripheral lymphocytes of tumor patients

    International Nuclear Information System (INIS)

    Klein, W.; Alth, G.; Klein, H.; Koren, H.

    1979-07-01

    The influence of radiotherapy and chemotherapy, respectively, on DNA excision repair was investigated in lymphocytes of the peripheral blood of 10 and 5 patients with malignancies. No effects on DNA repair were found using only betatrone of 60 Co-irradiation under normal conditions. Combination of both irradiation schedules over a longer period of therapy provoked an inhibition of DNA repair. Chemotherapy inhibits DNA repair immediately after starting therapy, but after relatively short time, the extent of DNA repair increases above normal level. (author)

  19. Optimization of loop-mediated isothermal amplification (LAMP) assays for the detection of Leishmania DNA in human blood samples.

    Science.gov (United States)

    Abbasi, Ibrahim; Kirstein, Oscar D; Hailu, Asrat; Warburg, Alon

    2016-10-01

    Visceral leishmaniasis (VL), one of the most important neglected tropical diseases, is caused by Leishmania donovani eukaryotic protozoan parasite of the genus Leishmania, the disease is prevalent mainly in the Indian sub-continent, East Africa and Brazil. VL can be diagnosed by PCR amplifying ITS1 and/or kDNA genes. The current study involved the optimization of Loop-mediated isothermal amplification (LAMP) for the detection of Leishmania DNA in human blood or tissue samples. Three LAMP systems were developed; in two of those the primers were designed based on shared regions of the ITS1 gene among different Leishmania species, while the primers for the third LAMP system were derived from a newly identified repeated region in the Leishmania genome. The LAMP tests were shown to be sufficiently sensitive to detect 0.1pg of DNA from most Leishmania species. The green nucleic acid stain SYTO16, was used here for the first time to allow real-time monitoring of LAMP amplification. The advantage of real time-LAMP using SYTO 16 over end-point LAMP product detection is discussed. The efficacy of the real time-LAMP tests for detecting Leishmania DNA in dried blood samples from volunteers living in endemic areas, was compared with that of qRT-kDNA PCR. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Different patterns of HIV-1 DNA after therapy discontinuation

    Directory of Open Access Journals (Sweden)

    Ghinelli Florio

    2005-09-01

    Full Text Available Abstract Background By persisting in infected cells for a long period of time, proviral HIV-1 DNA can represent an alternative viral marker to RNA viral load during the follow-up of HIV-1 infected individuals. In the present study sequential blood samples of 10 patients under antiretroviral treatment from 1997 with two NRTIs, who refused to continue any antiviral regimen, were analyzed for 16 – 24 weeks to study the possible relationship between DNA and RNA viral load. Methods The amount of proviral DNA was quantified by SYBR green real-time PCR in peripheral blood mononuclear cells from a selected group of ten patients with different levels of plasmatic viremia (RNA viral load. Results Variable levels of proviral DNA were found without any significant correlation between proviral load and plasma HIV-1 RNA levels. Results obtained showed an increase or a rebound in viral DNA in most patients, suggesting that the absence of therapy reflects an increase and/or a persistence of cells containing viral DNA. Conclusion Even though plasma HIV RNA levels remain the basic parameter to monitor the intensity of viral replication, the results obtained seem to indicate that DNA levels could represent an adjunct prognostic marker in monitoring HIV-1 infected subjects.

  1. New optimized DNA extraction protocol for fingerprints deposited on a special self-adhesive security seal and other latent samples used for human identification.

    Science.gov (United States)

    Kopka, Julieta; Leder, Monika; Jaureguiberry, Stella M; Brem, Gottfried; Boselli, Gabriel O

    2011-09-01

    Obtaining complete short tandem repeat (STR) profiles from fingerprints containing minimal amounts of DNA, using standard extraction techniques, can be difficult. The aim of this study was to evaluate a new kit, Fingerprint DNA Finder (FDF Kit), recently launched for the extraction of DNA and STR profiling from fingerprints placed on a special device known as Self-Adhesive Security Seal Sticker(®) and other latent fingerprints on forensic evidentiary material like metallic guns. The DNA extraction system is based on a reversal of the silica principle, and all the potential inhibiting substances are retained on the surface of a special adsorbent, while nucleic acids are not bound and remain in solution dramatically improving DNA recovery. DNA yield was quite variable among the samples tested, rendering in most of the cases (>90%) complete STR profiles, free of PCR inhibitors, and devoid of artifacts. Even samples with DNA amount below 100 pg could be successfully analyzed. © 2011 American Academy of Forensic Sciences.

  2. Detection of African Swine Fever Virus DNA in Blood Samples Stored on FTA Cards from Asymptomatic Pigs in Mbeya Region, Tanzania

    DEFF Research Database (Denmark)

    Braae, U. C.; Johansen, M. V.; Ngowi, H. A.

    2015-01-01

    The aim of the study was to assess whether blood samples collected onto FTA® cards could be used in combination with real-time PCR for the detection of African swine fever virus (ASFV) DNA in samples from resource-poor settings under the assumption that asymptomatically (sub-clinically) infected...... pigs may be present. Blood samples were collected from clinically healthy pigs from Mbeya Region, Tanzania. The blood samples were stored on FTA® cards and analysed by real-time PCR assays in duplicate; three pigs had high levels of viral DNA (Ct values of 27-29), and three pigs had a low level....../1) or a non-pathogenic (OURT T88/3) isolate of ASFV were collected, stored on FTA® cards and analysed in the same way. The blood from pigs infected with the OURT T88/1 isolate showed high levels of viral DNA (Ct 22-33), whereas infection with non-pathogenic OURT T88/3 isolate resulted in only low levels...

  3. Evaluating ethanol-based sample preservation to facilitate use of DNA barcoding in routine freshwater biomonitoring programs using benthic macroinvertebrates.

    Directory of Open Access Journals (Sweden)

    Eric D Stein

    Full Text Available Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI. On average, we obtained successful COI sequences (i.e. either full or partial barcodes for between 93-99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.

  4. Energetics of DNA repair: effects of temperature on DNA repair in UV-irradiated peripheral blood leucocytes from chronic myeloid leukemic patients

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, A.; Sharma, R.; Jain, V.K.

    1988-05-01

    The effects of different temperatures (34-43/sup 0/C) were studied on the repair of UV-induced (254-nm) DNA damage and its energetics in peripheral blood leucocytes of chronic myeloid leukaemic patients. DNA repair was measured by the unscheduled DNA synthesis (UDS) technique. Cellular energy supply was modulated by inhibitors of oxidative phosphorylation (antimycin-A) and glycolysis (2-deoxy-D-glucose). It was observed that there is an increase in the amount of DNA repair with increasing temperatures up to 40/sup 0/C and a fall thereafter. Longer periods of heat treatment (4 h) beyond 40/sup 0/C were observed to further decrease the DNA repair. Increasing temperatures were observed to have no significant effect on the parameters of energy metabolism. Further, the activation energy of DNA repair was calculated as 92 +- 46 kJ/mol (22 +- 11 kcal/mol), which did not alter significantly even in the presence of inhibitors of energy metabolism.

  5. Forensic genetic SNP typing of low-template DNA and highly degraded DNA from crime case samples

    DEFF Research Database (Denmark)

    Børsting, Claus; Mogensen, Helle Smidt; Morling, Niels

    2013-01-01

    the heterozygote balance. Allele drop-ins were only observed in experiments with 25 pg of DNA and not in experiments with 50 and 100 pg of DNA. The allele drop-in rate in the 25 pg experiments was 0.06% or 100 times lower than what was previously reported for STR typing of LtDNA. A composite model and two......Heterozygote imbalances leading to allele drop-outs and disproportionally large stutters leading to allele drop-ins are known stochastic phenomena related to STR typing of low-template DNA (LtDNA). The large stutters and the many drop-ins in typical STR stutter positions are artifacts from the PCR...... amplification of tandem repeats. These artifacts may be avoided by typing bi-allelic markers instead of STRs. In this work, the SNPforID multiplex assay was used to type LtDNA. A sensitized SNP typing protocol was introduced, that increased signal strengths without increasing noise and without affecting...

  6. Prognostic role of APC and RASSF1A promoter methylation status in cell free circulating DNA of operable gastric cancer patients.

    Science.gov (United States)

    Balgkouranidou, I; Matthaios, D; Karayiannakis, A; Bolanaki, H; Michailidis, P; Xenidis, N; Amarantidis, K; Chelis, L; Trypsianis, G; Chatzaki, E; Lianidou, E S; Kakolyris, S

    2015-08-01

    Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p APC promoter and elevated CEA (p = 0.033) as well as CA-19.9 (p = 0.032) levels, was noticed. The Kaplan-Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p = 0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Genotyping for DQA1 and PM loci in urine using PCR-based amplification: effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing.

    Science.gov (United States)

    Vu, N T; Chaturvedi, A K; Canfield, D V

    1999-05-31

    Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for individualization of biological samples have reached a new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investigated the influence of storage conditions, sample volume, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based genotyping for DQA1 and PM loci in urine. Urine specimens from male and female volunteers were divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by dialfiltration, using 3000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin method gave comparable typing results. Larger sample volume yielded a higher amount of DNA, but the typing rates were not affected for sample volumes between 1 and 5 ml. The quantifiable amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage samples produced significantly higher success rates than that of frozen samples, while there was only marginal difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of DQA1 + PM genotype was achieved for all samples of fresh urine, independent of gender

  8. Evaluation of PCR procedures for detecting and quantifying Leishmania donovani DNA in large numbers of dried human blood samples from a visceral leishmaniasis focus in Northern Ethiopia.

    Science.gov (United States)

    Abbasi, Ibrahim; Aramin, Samar; Hailu, Asrat; Shiferaw, Welelta; Kassahun, Aysheshm; Belay, Shewaye; Jaffe, Charles; Warburg, Alon

    2013-03-27

    Visceral Leishmaniasis (VL) is a disseminated protozoan infection caused by Leishmania donovani parasites which affects almost half a million persons annually. Most of these are from the Indian sub-continent, East Africa and Brazil. Our study was designed to elucidate the role of symptomatic and asymptomatic Leishmania donovani infected persons in the epidemiology of VL in Northern Ethiopia. The efficacy of quantitative real-time kinetoplast DNA/PCR (qRT-kDNA PCR) for detecting Leishmania donovani in dried-blood samples was assessed in volunteers living in an endemic focus. Of 4,757 samples, 680 (14.3%) were found positive for Leishmania k-DNA but most of those (69%) had less than 10 parasites/ml of blood. Samples were re-tested using identical protocols and only 59.3% of the samples with 10 parasite/ml or less were qRT-kDNA PCR positive the second time. Furthermore, 10.8% of the PCR negative samples were positive in the second test. Most samples with higher parasitemias remained positive upon re-examination (55/59 =93%). We also compared three different methods for DNA preparation. Phenol-chloroform was more efficient than sodium hydroxide or potassium acetate. DNA sequencing of ITS1 PCR products showed that 20/22 samples were Leishmania donovani while two had ITS1 sequences homologous to Leishmania major. Although qRT-kDNA PCR is a highly sensitive test, the dependability of low positives remains questionable. It is crucial to correlate between PCR parasitemia and infectivity to sand flies. While optimal sensitivity is achieved by targeting k-DNA, it is important to validate the causative species of VL by DNA sequencing.

  9. Microdosimetry calculations for monoenergetic electrons using Geant4-DNA combined with a weighted track sampling algorithm.

    Science.gov (United States)

    Famulari, Gabriel; Pater, Piotr; Enger, Shirin A

    2017-07-07

    The aim of this study was to calculate microdosimetric distributions for low energy electrons simulated using the Monte Carlo track structure code Geant4-DNA. Tracks for monoenergetic electrons with kinetic energies ranging from 100 eV to 1 MeV were simulated in an infinite spherical water phantom using the Geant4-DNA extension included in Geant4 toolkit version 10.2 (patch 02). The microdosimetric distributions were obtained through random sampling of transfer points and overlaying scoring volumes within the associated volume of the tracks. Relative frequency distributions of energy deposition f(>E)/f(>0) and dose mean lineal energy ([Formula: see text]) values were calculated in nanometer-sized spherical and cylindrical targets. The effects of scoring volume and scoring techniques were examined. The results were compared with published data generated using MOCA8B and KURBUC. Geant4-DNA produces a lower frequency of higher energy deposits than MOCA8B. The [Formula: see text] values calculated with Geant4-DNA are smaller than those calculated using MOCA8B and KURBUC. The differences are mainly due to the lower ionization and excitation cross sections of Geant4-DNA for low energy electrons. To a lesser extent, discrepancies can also be attributed to the implementation in this study of a new and fast scoring technique that differs from that used in previous studies. For the same mean chord length ([Formula: see text]), the [Formula: see text] calculated in cylindrical volumes are larger than those calculated in spherical volumes. The discrepancies due to cross sections and scoring geometries increase with decreasing scoring site dimensions. A new set of [Formula: see text] values has been presented for monoenergetic electrons using a fast track sampling algorithm and the most recent physics models implemented in Geant4-DNA. This dataset can be combined with primary electron spectra to predict the radiation quality of photon and electron beams.

  10. A misleading false-negative result using Neisseria gonorrhoeae opa MGB multiplex PCR assay in patient's rectal sample due to partial mutations of the opa gene.

    Science.gov (United States)

    Vahidnia, Ali; van Empel, Pieter Jan; Costa, Sandra; Oud, Rob T N; van der Straaten, Tahar; Bliekendaal, Harry; Spaargaren, Joke

    2015-07-01

    A 53-year-old homosexual man presented at his general practitioner (GP) practice with a suspicion of sexually transmitted infection. Initial NAAT screening was performed for Chlamydia trachomatis and Neisseria gonorrhoeae. The patient was positive for Neisseria gonorrhoeae both for his urine and rectal sample. The subsequent confirmation test for Neisseria gonorrhoeae by a second laboratory was only confirmed for the urine sample and the rectal sample was negative. We report a case of a potential false-negative diagnosis of Neisseria gonorrhoeae due to mutations of DNA sequence in the probe region of opa-MGB assay of the rectal sample. The patient did not suffer any discomfort as diagnosis of Neisseria gonorrhoeae in his urine sample had already led to treatment by prescribing the patient with Ceftriaxone 500 mg IV dissolved in 1 ml lidocaine 2% and 4 mL saline. The patient also received a prescription for Azithromycin (2x500 mg).

  11. Automated extraction of DNA from reference samples from various types of biological materials on the Qiagen BioRobot EZ1 Workstation

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Jørgensen, Mads; Hansen, Anders Johannes

    2009-01-01

    , and muscle biopsies. The DNA extraction was validated according to EN/ISO 17025 for the STR kits AmpFlSTR« Identifiler« and AmpFlSTR« Yfiler« (Applied Biosystems). Of 298 samples extracted, 11 (4%) did not yield acceptable results. In conclusion, we have demonstrated that extraction of DNA from various types......We have validated and implemented a protocol for DNA extraction from various types of biological materials using a Qiagen BioRobot EZ1 Workstation. The sample materials included whole blood, blood from deceased, buccal cells on Omni swabs and FTA Cards, blood on FTA Cards and cotton swabs...... of biological material can be performed quickly and without the use of hazardous chemicals, and that the DNA may be successfully STR typed according to the requirements of forensic genetic investigations accredited according to EN/ISO 17025...

  12. Prevalence of parvovirus B19 and parvovirus V9 DNA and antibodies in paired bone marrow and serum samples from healthy individuals.

    Science.gov (United States)

    Heegaard, Erik D; Petersen, Bodil Laub; Heilmann, Carsten J; Hornsleth, Allan

    2002-03-01

    Parvovirus B19 (hereafter referred to as B19) exhibits a marked tropism to human bone marrow (BM), and infection may lead to erythema infectiosum, arthropathy, hydrops fetalis, and various hematologic disorders. Recently, a distinct parvovirus isolate termed V9 with an unknown clinical spectrum was discovered. In contrast to the many studies of B19 serology and viremia, valid information on the frequency of B19 or V9 DNA in the BM of healthy individuals is limited. To develop a reference value, paired BM and serum samples from healthy subjects were tested for the presence of B19 and V9 DNA and specific antibodies. Immunoglobulin M (IgM) was not found in any of the serum samples. The prevalence of IgG showed a gradual and steady increase from 37% in children aged 1 to 5 years to 87% in people aged >50 years. When 190 well-characterized subjects were examined, B19 DNA was detected in the BM of 4 individuals (2.1%; 95% confidence interval, 0.58 to 5.3%) while none of the paired serum samples showed evidence of circulating viral DNA. V9 DNA was not found in any of the BM or serum samples. The finding of B19 DNA probably indicated a primary infection in one 7-year-old individual and reinfection or reactivation of persistent infection in the remaining three persons, aged 47 to 58 years. Serving as a benchmark for future studies, these findings are useful when interpreting epidemiologic data, performing BM transplantation, or considering clinical implications of parvovirus infection.

  13. Undetected human papillomavirus DNA and uterine cervical carcinoma. Association with cancer recurrence

    International Nuclear Information System (INIS)

    Okuma, Kae; Yamashita, Hideomi; Nakagawa, Keiichi; Yokoyama, Terufumi; Kawana, Kei

    2016-01-01

    The time course of human papillomavirus (HPV) DNA clearance was studied in patients with carcinoma of the cervix during follow-up after primary radical radiotherapy (RT). This study investigated the relationship between timing of HPV clearance and RT effectiveness. A total of 71 consecutive patients who were treated for cervical cancer with primary radical radiotherapy and high-dose rate intracavitary brachytherapy with or without chemotherapy were enrolled in the study. Samples for HPV DNA examination were taken before (1) treatment, (2) every brachytherapy, and (3) every follow-up examination. The times when HPV DNA was undetected were analyzed for association with recurrence-free survival. HPV DNA was not detected in 13 patients (18 %) before RT. Of the 58 patients with HPV DNA detected before treatment, HPV DNA was not detected in 34 % during treatment and in 66 % after the treatment. Within 6 months after RT, HPV DNA was detected in 0 % of all patients. The patients were followed up for a median period of 43 months (range 7-70 months). In all, 20 patients were found to develop recurrence. The 3-year cumulative disease-free survival (DFS) rate was 71 ± 5.4 % for all 71 patients. In multivariate analysis, DFS was significantly associated with HPV (detected vs. not detected) with a hazard ratio of 0.07 (95 % confidence interval 0.008-0.6, p = 0.009). In this study, patients in whom HPV was not detected had the worst prognosis. Six months after RT, HPV DNA was detected in 0 % of the patients. Patients in whom HPV DNA could not be detected before treatment need careful follow-up for recurrence and may be considered for additional, or alternative treatment. (orig.) [de

  14. Modeling the Sensitivity of Field Surveys for Detection of Environmental DNA (eDNA.

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    Martin T Schultz

    Full Text Available The environmental DNA (eDNA method is the practice of collecting environmental samples and analyzing them for the presence of a genetic marker specific to a target species. Little is known about the sensitivity of the eDNA method. Sensitivity is the probability that the target marker will be detected if it is present in the water body. Methods and tools are needed to assess the sensitivity of sampling protocols, design eDNA surveys, and interpret survey results. In this study, the sensitivity of the eDNA method is modeled as a function of ambient target marker concentration. The model accounts for five steps of sample collection and analysis, including: 1 collection of a filtered water sample from the source; 2 extraction of DNA from the filter and isolation in a purified elution; 3 removal of aliquots from the elution for use in the polymerase chain reaction (PCR assay; 4 PCR; and 5 genetic sequencing. The model is applicable to any target species. For demonstration purposes, the model is parameterized for bighead carp (Hypophthalmichthys nobilis and silver carp (H. molitrix assuming sampling protocols used in the Chicago Area Waterway System (CAWS. Simulation results show that eDNA surveys have a high false negative rate at low concentrations of the genetic marker. This is attributed to processing of water samples and division of the extraction elution in preparation for the PCR assay. Increases in field survey sensitivity can be achieved by increasing sample volume, sample number, and PCR replicates. Increasing sample volume yields the greatest increase in sensitivity. It is recommended that investigators estimate and communicate the sensitivity of eDNA surveys to help facilitate interpretation of eDNA survey results. In the absence of such information, it is difficult to evaluate the results of surveys in which no water samples test positive for the target marker. It is also recommended that invasive species managers articulate concentration

  15. Histopathological examination of nerve samples from pure neural leprosy patients: obtaining maximum information to improve diagnostic efficiency

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    Sérgio Luiz Gomes Antunes

    2012-03-01

    Full Text Available Nerve biopsy examination is an important auxiliary procedure for diagnosing pure neural leprosy (PNL. When acid-fast bacilli (AFB are not detected in the nerve sample, the value of other nonspecific histological alterations should be considered along with pertinent clinical, electroneuromyographical and laboratory data (the detection of Mycobacterium leprae DNA with polymerase chain reaction and the detection of serum anti-phenolic glycolipid 1 antibodies to support a possible or probable PNL diagnosis. Three hundred forty nerve samples [144 from PNL patients and 196 from patients with non-leprosy peripheral neuropathies (NLN] were examined. Both AFB-negative and AFB-positive PNL samples had more frequent histopathological alterations (epithelioid granulomas, mononuclear infiltrates, fibrosis, perineurial and subperineurial oedema and decreased numbers of myelinated fibres than the NLN group. Multivariate analysis revealed that independently, mononuclear infiltrate and perineurial fibrosis were more common in the PNL group and were able to correctly classify AFB-negative PNL samples. These results indicate that even in the absence of AFB, these histopathological nerve alterations may justify a PNL diagnosis when observed in conjunction with pertinent clinical, epidemiological and laboratory data.

  16. Mitochondrial DNA copy number threshold in mtDNA depletion myopathy.

    Science.gov (United States)

    Durham, S E; Bonilla, E; Samuels, D C; DiMauro, S; Chinnery, P F

    2005-08-09

    The authors measured the absolute amount of mitochondrial DNA (mtDNA) within single muscle fibers from two patients with thymidine kinase 2 (TK2) deficiency and two healthy controls. TK2 deficient fibers containing more than 0.01 mtDNA/microm3 had residual cytochrome c oxidase (COX) activity. This defines the minimum amount of wild-type mtDNA molecules required to maintain COX activity in skeletal muscle and provides an explanation for the mosaic histochemical pattern seen in patients with mtDNA depletion syndrome.

  17. Effect of Clozapine on DNA Methylation in Peripheral Leukocytes from Patients with Treatment-Resistant Schizophrenia

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    Makoto Kinoshita

    2017-03-01

    Full Text Available Clozapine is an atypical antipsychotic, that is established as the treatment of choice for treatment-resistant schizophrenia (SCZ. To date, no study investigating comprehensive DNA methylation changes in SCZ patients treated with chronic clozapine has been reported. The purpose of the present study is to reveal the effects of clozapine on DNA methylation in treatment-resistant SCZ. We conducted a genome-wide DNA methylation profiling in peripheral leukocytes (485,764 CpG dinucleotides from treatment-resistant SCZ patients treated with clozapine (n = 21 in a longitudinal study. Significant changes in DNA methylation were observed at 29,134 sites after one year of treatment with clozapine, and these genes were enriched for “cell substrate adhesion” and “cell matrix adhesion” gene ontology (GO terms. Furthermore, DNA methylation changes in the CREBBP (CREB binding protein gene were significantly correlated with the clinical improvements. Our findings provide insights into the action of clozapine in treatment-resistant SCZ.

  18. Detection of multidrug-resistant tuberculosis from stored DNA Samples: A multicenter study

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    Marie Sylvianne Rabodoarivelo

    2018-01-01

    Full Text Available Background: In low-income countries, rapid detection of tuberculosis (TB drug resistance is often restricted by the difficulties of transporting and storing sputum samples from remote health centers to the reference laboratories where molecular tests are available. The aim of this study was to evaluate the performance of four transport and storage systems for molecular detection of rifampicin (RIF and isoniazid (INH resistance. Methods: This was a multicenter study. Molecular detection of RIF and INH resistance was performed directly from smear-positive TB sputa spotted on a slide, FTA card, GenoCard, and ethanol using the Genotype MTBDRplus assay. The performance of the DNA extraction method from each storage support to detect drug resistance was assessed by calculating their sensitivity and specificity compared to the phenotypic method. Results: From all sites, the overall sensitivity and specificity for RIF-resistance detection was 88% and 85%, respectively, for slides, 86% and 92%, respectively, for GenoCard, 87% and 89%, respectively, for FTA card, and 88% and 92%, respectively, for ethanol. For INH-resistance detection, the overall sensitivity and specificity was 82% and 90%, respectively, for slides, 85% and 96%, respectively, for GenoCard, 86% and 92%, respectively, for FTA card, and 86% and 94%, respectively, for ethanol. Conclusion: Smear slides and filter cards showed to be very useful tools to facilitate DNA extraction from sputum samples with the potential to accelerate the detection of drug resistance in remote areas.

  19. Detection of multidrug-resistant tuberculosis from stored DNA Samples: A multicenter study.

    Science.gov (United States)

    Rabodoarivelo, Marie Sylvianne; Brandao, A; Cergole Novella, M C; C Bombonatte, A G; Imperiale, B; Rakotosamimanana, N; Morcillo, N; Rasolofo, V; Palomino, J C; Martin, A

    2018-01-01

    In low-income countries, rapid detection of tuberculosis (TB) drug resistance is often restricted by the difficulties of transporting and storing sputum samples from remote health centers to the reference laboratories where molecular tests are available. The aim of this study was to evaluate the performance of four transport and storage systems for molecular detection of rifampicin (RIF) and isoniazid (INH) resistance. This was a multicenter study. Molecular detection of RIF and INH resistance was performed directly from smear-positive TB sputa spotted on a slide, FTA card, GenoCard, and ethanol using the Genotype MTBDRplus assay. The performance of the DNA extraction method from each storage support to detect drug resistance was assessed by calculating their sensitivity and specificity compared to the phenotypic method. From all sites, the overall sensitivity and specificity for RIF-resistance detection was 88% and 85%, respectively, for slides, 86% and 92%, respectively, for GenoCard, 87% and 89%, respectively, for FTA card, and 88% and 92%, respectively, for ethanol. For INH-resistance detection, the overall sensitivity and specificity was 82% and 90%, respectively, for slides, 85% and 96%, respectively, for GenoCard, 86% and 92%, respectively, for FTA card, and 86% and 94%, respectively, for ethanol. Smear slides and filter cards showed to be very useful tools to facilitate DNA extraction from sputum samples with the potential to accelerate the detection of drug resistance in remote areas.

  20. Oral antioxidant treatment partly improves integrity of human sperm DNA in infertile grade I varicocele patients.

    Science.gov (United States)

    Gual-Frau, Josep; Abad, Carlos; Amengual, María J; Hannaoui, Naim; Checa, Miguel A; Ribas-Maynou, Jordi; Lozano, Iris; Nikolaou, Alexandros; Benet, Jordi; García-Peiró, Agustín; Prats, Juan

    2015-09-01

    Infertile males with varicocele have the highest percentage of sperm cells with damaged DNA, compared to other infertile groups. Antioxidant treatment is known to enhance the integrity of sperm DNA; however, there are no data on the effects in varicocele patients. We thus investigated the potential benefits of antioxidant treatment specifically in grade I varicocele males. Twenty infertile patients with grade I varicocele were given multivitamins (1500 mg L-Carnitine, 60 mg vitamin C, 20 mg coenzyme Q10, 10 mg vitamin E, 200 μg vitamin B9, 1 μg vitamin B12, 10 mg zinc, 50 μg selenium) daily for three months. Semen parameters including total sperm count, concentration, progressive motility, vitality, and morphology were determined before and after treatment. In addition, sperm DNA fragmentation and the amount of highly degraded sperm cells were analyzed by Sperm Chromatin Dispersion. After treatment, patients showed an average relative reduction of 22.1% in sperm DNA fragmentation (p = 0.02) and had 31.3% fewer highly degraded sperm cells (p = 0.07). Total numbers of sperm cells were increased (p = 0.04), but other semen parameters were unaffected. These data suggest that sperm DNA integrity in grade I varicocele patients may be improved by oral antioxidant treatment.

  1. Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma

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    Hagen Jeffrey A

    2007-10-01

    Full Text Available Abstract Background Lung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients. Results We identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value Conclusion The identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.

  2. Molecular species identification with rich floristic sampling: DNA barcoding the pteridophyte flora of Japan.

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    Atsushi Ebihara

    Full Text Available BACKGROUND: DNA barcoding is expected to be an effective identification tool for organisms with heteromorphic generations such as pteridophytes, which possess a morphologically simple gametophyte generation. Although a reference data set including complete coverage of the target local flora/fauna is necessary for accurate identification, DNA barcode studies including such rich taxonomic sampling on a countrywide scale are lacking. METHODOLOGY/PRINCIPAL FINDINGS: The Japanese pteridophyte flora (733 taxa including subspecies and varieties was used to test the utility of two plastid DNA barcode regions (rbcL and trnH-psbA with the intention of developing an identification system for native gametophytes. DNA sequences were obtained from each of 689 (94.0% taxa for rbcL and 617 (84.2% taxa for trnH-psbA. Mean interspecific divergence values across all taxon pairs (K2P genetic distances did not reveal a significant difference in rate between trnH-psbA and rbcL, but mean K2P distances of each genus showed significant heterogeneity according to systematic position. The minimum fail rate of taxon discrimination in an identification test using BLAST (12.52% was obtained when rbcL and trnH-psbA were combined, and became lower in datasets excluding infraspecific taxa or apogamous taxa, or including sexual diploids only. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the overall effectiveness of DNA barcodes for species identification in the Japanese pteridophyte flora. Although this flora is characterized by a high occurrence of apogamous taxa that pose a serious challenge to identification using DNA barcodes, such taxa are limited to a small number of genera, and only minimally detract from the overall success rate. In the case that a query sequence is matched to a known apogamous genus, routine species identification may not be possible. Otherwise, DNA barcoding is a practical tool for identification of most Japanese pteridophytes, and is especially

  3. Nuclear DNA as Predictor of Acute Kidney Injury in Patients Undergoing Coronary Artery Bypass Graft: A Pilot Study.

    Science.gov (United States)

    Likhvantsev, Valery V; Landoni, Giovanni; Grebenchikov, Oleg A; Skripkin, Yuri V; Zabelina, Tatiana S; Zinovkina, Liudmila A; Prikhodko, Anastasia S; Lomivorotov, Vladimir V; Zinovkin, Roman A

    2017-12-01

    To measure the release of plasma nuclear deoxyribonucleic acid (DNA) and to assess the relationship between nuclear DNA level and acute kidney injury occurrence in patients undergoing cardiac surgery. Cardiovascular anesthesiology and intensive care unit of a large tertiary-care university hospital. Prospective observational study. Fifty adult patients undergoing cardiac surgery. Nuclear DNA concentration was measured in the plasma. The relationship between the level of nuclear DNA and the incidence of acute kidney injury after coronary artery bypass grafting was investigated. Cardiac surgery leads to significant increase in plasma nuclear DNA with peak levels 12 hours after surgery (median [interquartile range] 7.0 [9.6-22.5] µg/mL). No difference was observed between off-pump and on-pump surgical techniques. Nuclear DNA was the only predictor of acute kidney injury between baseline and early postoperative risk factors. The authors found an increase of nuclear DNA in the plasma of patients who had undergone coronary artery bypass grafting, with a peak after 12 hours and an association of nuclear DNA with postoperative acute kidney injury. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

    Science.gov (United States)

    Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

    2014-11-01

    There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss

  5. Presence of human papilloma virus, herpes simplex virus and Epstein-Barr virus DNA in oral biopsies from Sudanese patients with regard to toombak use.

    Science.gov (United States)

    Jalouli, Jamshid; Ibrahim, Salah O; Sapkota, Dipak; Jalouli, Miranda M; Vasstrand, Endre N; Hirsch, Jan M; Larsson, Per-Anders

    2010-09-01

    Using PCR/DNA sequencing, we investigated the prevalence of human papillomavirus (HPV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) DNA in brush biopsies obtained from 150 users of Sudanese snuff (toombak) and 25 non-users of toombak in formalin-fixed paraffin-embedded tissue samples obtained from 31 patients with oral dysplasias (25 toombak users and 6 non-users), and from 217 patients with oral cancers (145 toombak users and 72 non-users). In the brush tissue samples from toombak users, HPV was detected in 60 (40%), HSV in 44 (29%) and EBV in 97 (65%) of the samples. The corresponding figures for the 25 samples from non-users were 17 (68%) positive for HPV, 6 (24%) positive for HSV and 21 (84%) for EBV. The formalin-fixed samples with oral dysplasias were all negative for HPV. In the 145 oral cancer samples from toombak users, HPV was detected in 39 (27%), HSV in 15 (10%) and EBV in 53 (37%) of the samples. The corresponding figures for the samples from non-users were 15 (21%) positive for HPV, 5 (7%) for HSV and 16 (22%) for EBV. These findings illustrate that prevalence of HSV, HPV and EBV infections are common and may influence oral health and cancer development. It is not obvious that cancer risk is increased in infected toombak users. These observations warrant further studies involving toombak-associated oral lesions, to uncover the possible mechanisms of these viral infections in the development of oral cancer, and the influence of toombak on these viruses. © 2010 John Wiley & Sons A/S.

  6. SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients.

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    Julia Stadler

    Full Text Available Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent "gold standard". Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution, at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.

  7. Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome.

    Science.gov (United States)

    Huang, Xiao-Ying; Li, Hong; Xu, Xiao-Mei; Wang, Liang-Xing

    2014-08-01

    The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102 healthy adults were assessed to determine the levels of fasting blood glucose, total cholesterol, triglyceride (TG) and high-density and low-density lipoproteins (LDL). Furthermore, peripheral mtDNA was extracted and bidirectional sequencing was conducted to enable mutation screening. In the mtDNA D-loop region, 178 mutation sites were identified, of which 115 sites were present in the two groups. The number of non-common sites in the OSAHS group was significantly higher compared with the control group (P0.05). A total of 21 cases in the severe OSAHS group exhibited mutation rates of >10%. In the control group, there were 24 cases where the np73A-G and np263A-G mutations were predominant. The np303-np315 region was identified to be the highly variable region and various mutation forms were observed. Statistically significant differences were observed in the neck perimeter, TG and LDL levels among the OSAHS-no-mutation subgroups (P<0.05) and LDL was shown to be associated with an mtDNA mutation in the OSAHS group. Numerous polymorphic mutation sites were identified in the mtDNA D-loop region of the OSAHS group. Therefore, mtDNA mutation sites may be closely associated with the clinical manifestations and complications of OSAHS.

  8. Performance of Identifiler Direct and PowerPlex 16 HS on the Applied Biosystems 3730 DNA Analyzer for processing biological samples archived on FTA cards.

    Science.gov (United States)

    Laurin, Nancy; DeMoors, Anick; Frégeau, Chantal

    2012-09-01

    Direct amplification of STR loci from biological samples collected on FTA cards without prior DNA purification was evaluated using Identifiler Direct and PowerPlex 16 HS in conjunction with the use of a high throughput Applied Biosystems 3730 DNA Analyzer. In order to reduce the overall sample processing cost, reduced PCR volumes combined with various FTA disk sizes were tested. Optimized STR profiles were obtained using a 0.53 mm disk size in 10 μL PCR volume for both STR systems. These protocols proved effective in generating high quality profiles on the 3730 DNA Analyzer from both blood and buccal FTA samples. Reproducibility, concordance, robustness, sample stability and profile quality were assessed using a collection of blood and buccal samples on FTA cards from volunteer donors as well as from convicted offenders. The new developed protocols offer enhanced throughput capability and cost effectiveness without compromising the robustness and quality of the STR profiles obtained. These results support the use of these protocols for processing convicted offender samples submitted to the National DNA Data Bank of Canada. Similar protocols could be applied to the processing of casework reference samples or in paternity or family relationship testing. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  9. Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

    Science.gov (United States)

    Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

    2016-11-01

    Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  10. DNA-repair and mutations in immuncompetent cells from patients with rheumatic diseases and corresponding animal models

    International Nuclear Information System (INIS)

    Altmann, H.

    1977-01-01

    Unscheduled DNA synthesis was investigated in lymphocytes of patients with different inflammatory rheumatic diseases. After γ-irradiation H 3 -thymidin incorporation in DNA and DNA rejoining was reduced. After UV-irradiation the first step (90 min) of unscheduled DNA synthesis was above the controls. Some animal models for human diseases showed the same trend. An infectious ethiology was discussed for some of these diseases. (author)

  11. Oxidative DNA damage in lung tissue from patients with COPD is clustered in functionally significant sequences

    Directory of Open Access Journals (Sweden)

    Viktor M Pastukh

    2011-03-01

    Full Text Available Viktor M Pastukh1, Li Zhang2, Mykhaylo V Ruchko1, Olena Gorodnya1, Gina C Bardwell1, Rubin M Tuder2, Mark N Gillespie11Department of Pharmacology and Center for Lung Biology, University of South Alabama College of Medicine, Mobile, AL, USA; 2Program in Translational Lung Research, Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado at Denver, Aurora, CO, USAAbstract: Lung tissue from COPD patients displays oxidative DNA damage. The present study determined whether oxidative DNA damage was randomly distributed or whether it was localized in specific sequences in either the nuclear or mitochondrial genomes. The DNA damage-specific histone, gamma-H2AX, was detected immunohistochemically in alveolar wall cells in lung tissue from COPD patients but not control subjects. A PCR-based method was used to search for oxidized purine base products in selected 200 bp sequences in promoters and coding regions of the VEGF, TGF-β1, HO-1, Egr1, and β-actin genes while quantitative Southern blot analysis was used to detect oxidative damage to the mitochondrial genome in lung tissue from control subjects and COPD patients. Among the nuclear genes examined, oxidative damage was detected in only 1 sequence in lung tissue from COPD patients: the hypoxic response element (HRE of the VEGF promoter. The content of VEGF mRNA also was reduced in COPD lung tissue. Mitochondrial DNA content was unaltered in COPD lung tissue, but there was a substantial increase in mitochondrial DNA strand breaks and/or abasic sites. These findings show that oxidative DNA damage in COPD lungs is prominent in the HRE of the VEGF promoter and in the mitochondrial genome and raise the intriguing possibility that genome and sequence-specific oxidative DNA damage could contribute to transcriptional dysregulation and cell fate decisions in COPD.Keywords: DNA damage, VEGF hypoxic response element, mtDNA, COPD

  12. Establishing the UK DNA Bank for motor neuron disease (MND).

    Science.gov (United States)

    Smith, Lucy; Cupid, B C; Dickie, B G M; Al-Chalabi, A; Morrison, K E; Shaw, C E; Shaw, P J

    2015-07-14

    In 2003 the Motor Neurone Disease (MND) Association, together with The Wellcome Trust, funded the creation of a national DNA Bank specific for MND. It was anticipated that the DNA Bank would constitute an important resource to researchers worldwide and significantly increase activity in MND genetic research. The DNA Bank houses over 3000 high quality DNA samples, all of which were donated by people living with MND, family members and non-related controls, accompanied by clinical phenotype data about the patients. Today the primary focus of the UK MND DNA Bank still remains to identify causative and disease modifying factors for this devastating disease.

  13. DNA polymerase zeta cooperates with polymerases kappa and iota in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients.

    Science.gov (United States)

    Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

    2009-07-14

    Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase eta, the POLH gene product. A deficiency in DNA polymerase eta due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase zeta cooperates with DNA polymerases kappa and iota to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases zeta and kappa, but not iota, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.

  14. DNA polymerase ζ cooperates with polymerases κ and ι in translesion DNA synthesis across pyrimidine photodimers in cells from XPV patients

    Science.gov (United States)

    Ziv, Omer; Geacintov, Nicholas; Nakajima, Satoshi; Yasui, Akira; Livneh, Zvi

    2009-01-01

    Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase η, the POLH gene product. A deficiency in DNA polymerase η due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase ζ cooperates with DNA polymerases κ and ι to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases ζ and κ, but not ι, protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load. PMID:19564618

  15. Mitochondrial DNA damage and vascular function in patients with diabetes mellitus and atherosclerotic cardiovascular disease.

    Science.gov (United States)

    Fetterman, Jessica L; Holbrook, Monica; Westbrook, David G; Brown, Jamelle A; Feeley, Kyle P; Bretón-Romero, Rosa; Linder, Erika A; Berk, Brittany D; Weisbrod, Robert M; Widlansky, Michael E; Gokce, Noyan; Ballinger, Scott W; Hamburg, Naomi M

    2016-03-31

    Prior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease. We assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochondrial DNA damage levels (β = 0.14 ± 0.13, P = 0.04 and β = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochondrial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function. We found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical atherosclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact.

  16. The correlation between DNA ploidy and the clinicohistologic findings in colorectal cancer

    International Nuclear Information System (INIS)

    Lee, Suk Ho; Kim, Hun Jung; Kim, Woo Chul; Cho, Young Kap; Loh, John J. K.; Woo, Ze Hong; Hwang, Tae Sook

    2000-01-01

    DNA ploidy pattern was shown to correlate with several clinicohistologic findings in several tumors. Aim of this study was to evaluate the correlation of the clinicohistologic findings in colorectal cancer and the failure pattern in rectosigmoid cancer with DNA ploidy. DNA flow cytometry using the Hedley methods on paraffin embedded specimen from 117 patients with colorectal cancers after curative resection was performed. We tried to find the correlation between DNA ploidy and various clinicohistologic findings. And then the correlation DNA ploidy and the failure pattern in 75 patients of rectosigmoid cancer was analized. Forty samples (34.2%) from tumors gave aneuploidy histogram. There was no significant difference in the frequency of DNA aneuploidy in terms of age, sex, depth of invasion, location and Dukes stage. But there was a significant correlation between DNA ploidy and the failure rates in Dukes stage B rectosigmoid cancer (p=0.048). These findings suggest that DNA ploidy pattern shows the correlation with the treatment failure rates in Dukes stage B rectosigmoid, but not with many other clinicohistologic findings. However, more patients will be needed to disclose these findings

  17. Molecular-based rapid inventories of sympatric diversity: a comparison of DNA barcode clustering methods applied to geography-based vs clade-based sampling of amphibians.

    Science.gov (United States)

    Paz, Andrea; Crawford, Andrew J

    2012-11-01

    Molecular markers offer a universal source of data for quantifying biodiversity. DNA barcoding uses a standardized genetic marker and a curated reference database to identify known species and to reveal cryptic diversity within wellsampled clades. Rapid biological inventories, e.g. rapid assessment programs (RAPs), unlike most barcoding campaigns, are focused on particular geographic localities rather than on clades. Because of the potentially sparse phylogenetic sampling, the addition of DNA barcoding to RAPs may present a greater challenge for the identification of named species or for revealing cryptic diversity. In this article we evaluate the use of DNA barcoding for quantifying lineage diversity within a single sampling site as compared to clade-based sampling, and present examples from amphibians. We compared algorithms for identifying DNA barcode clusters (e.g. species, cryptic species or Evolutionary Significant Units) using previously published DNA barcode data obtained from geography-based sampling at a site in Central Panama, and from clade-based sampling in Madagascar. We found that clustering algorithms based on genetic distance performed similarly on sympatric as well as clade-based barcode data, while a promising coalescent-based method performed poorly on sympatric data. The various clustering algorithms were also compared in terms of speed and software implementation. Although each method has its shortcomings in certain contexts, we recommend the use of the ABGD method, which not only performs fairly well under either sampling method, but does so in a few seconds and with a user-friendly Web interface.

  18. High-risk human papillomavirus (HPV screening and detection in healthy patient saliva samples: a pilot study

    Directory of Open Access Journals (Sweden)

    Wang Robert C

    2011-10-01

    Full Text Available Abstract Background The human papillomaviruses (HPV are a large family of non-enveloped DNA viruses, mainly associated with cervical cancers. Recent epidemiologic evidence has suggested that HPV may be an independent risk factor for oropharyngeal cancers. Evidence now suggests HPV may modulate the malignancy process in some tobacco- and alcohol-induced oropharynx tumors, but might also be the primary oncogenic factor for inducing carcinogenesis among some non-smokers. More evidence, however, is needed regarding oral HPV prevalence among healthy adults to estimate risk. The goal of this study was to perform an HPV screening of normal healthy adults to assess oral HPV prevalence. Methods Healthy adult patients at a US dental school were selected to participate in this pilot study. DNA was isolated from saliva samples and screened for high-risk HPV strains HPV16 and HPV18 and further processed using qPCR for quantification and to confirm analytical sensitivity and specificity. Results Chi-square analysis revealed the patient sample was representative of the general clinic population with respect to gender, race and age (p Conclusions The successful recruitment and screening of healthy adult patients revealed HPV16, but not HPV18, was present in a small subset. These results provide new information about oral HPV status, which may help to contextualize results from other studies that demonstrate oral cancer rates have risen in the US among both females and minorities and in some geographic areas that are not solely explained by rates of tobacco and alcohol use. The results of this study may be of significant value to further our understanding of oral health and disease risk, as well as to help design future studies exploring the role of other factors that influence oral HPV exposure, as well as the short- and long-term consequences of oral HPV infection.

  19. MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

    Science.gov (United States)

    McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

    2016-06-01

    Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

  20. Quantitation of DNA adducts by stable isotope dilution mass spectrometry

    Science.gov (United States)

    Tretyakova, Natalia; Goggin, Melissa; Janis, Gregory

    2012-01-01

    Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations potentially contributing to the development of cancer. Due to their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples – including immunoassay, HPLC, and 32P-postlabeling – isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adducts concentrations in biological samples are between 0.01 – 10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry – especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS – have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke. PMID:22827593

  1. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Verónica Loera-Castañeda

    2014-01-01

    Full Text Available Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS. Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12% harbored the A8027G polymorphism and three of them were early onset (EO AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn’t been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.

  2. Circulating Tumor DNA Analysis for Liver Cancers and Its Usefulness as a Liquid BiopsySummary

    Directory of Open Access Journals (Sweden)

    Atsushi Ono

    2015-09-01

    Full Text Available Background & Aims: Circulating tumor DNA (ctDNA carrying tumor-specific sequence alterations has been found in the cell-free fraction of blood. Liver cancer tumor specimens are difficult to obtain, and noninvasive methods are required to assess cancer progression and characterize underlying genomic features. Methods: We analyzed 46 patients with hepatocellular carcinoma who underwent hepatectomy or liver transplantation and for whom whole-genome sequencing data was available. We designed personalized assays targeting somatic rearrangements of each tumor to quantify serum ctDNA. Exome sequencing was performed using cell-free DNA paired primary tumor tissue DNA from a patient with recurrent liver cancer after transcatheter arterial chemoembolization (TACE. Results: We successfully detected ctDNA from 100 μL of serum samples in 7 of the 46 patients before surgery, increasing with disease progression. The cumulative incidence of recurrence and extrahepatic metastasis in the ctDNA-positive group were statistically significantly worse than in the ctDNA-negative group (P = .0102 and .0386, respectively. Multivariate analysis identified ctDNA (OR 6.10; 95% CI, 1.11–33.33, P = .038 as an independent predictor of microscopic vascular invasion of the portal vein (VP. We identified 45 nonsynonymous somatic mutations in cell-free DNA after TACE and 71 nonsynonymous somatic mutations in primary tumor tissue by exome sequencing. We identified 25 common mutations in both samples, and 83% of mutations identified in the primary tumor could be detected in the cell-free DNA. Conclusions: The presence of ctDNA reflects tumor progression, and detection of ctDNA can predict VP and recurrence, especially extrahepatic metastasis within 2 years. Our study demonstrated the usefulness of ctDNA detection and sequencing analysis of cell-free DNA for personalized treatment of liver cancer. Keywords: Circulating Tumor DNA, Exome Sequencing, Hepatocellular

  3. Detection of Mycobacterium leprae DNA for 36kDa protein in urine from leprosy patients: a preliminary report Detecção do DNA do Mycobacterium leprae para proteína 36 kDa na urina de pacientes com hanseníase: relato preliminar

    Directory of Open Access Journals (Sweden)

    Om Parkash

    2004-10-01

    Full Text Available We have searched for Mycobacterium leprae DNA for 36kDa protein in urine using a M. leprae specific PCR technique. A limited number of 16 patients (of which 11 belonged to lepromatous leprosy and five to tuberculoid leprosy and eight healthy individuals were included for the present study. The number of urine samples positive by PCR were 36.4% (4/11 in lepromatous patients and 40% (2/5 in tuberculoid patients. None of the samples from healthy individuals was positive. To our knowledge, the results indicate, for the first time, the presence of M. leprae DNA in urine from leprosy patients. Another important finding obtained out of the study is that amongst treated patients 66.6% (4/6 were positive whereas amongst untreated only 20% (2/10 were positive. From the present indicative data it appears that treatment improves the PCR results with urine as a sample. Thus, the approach could prove to be useful for monitoring the treatment response of individual patients and needs to be further evaluated with a large number of patients.Pesquisamos o DNA do Mycobacterium leprae para proteína 36 kDa na urina usando a técnica do PCR específica para M. leprae. Um número limitado de 16 pacientes (dos quais 11 tinham hanseníase multibacilar e cinco hanseníase paucibacilar e oito indivíduos saudáveis foram incluídos neste estudo. O número de amostras de urina positivas pelo PCR foi de 36,4% (4/11 em pacientes com hanseníase multibacilar e 40% (2/5 em pacientes com hanseníase paucibacilar. Nenhuma das amostras de indivíduos saudáveis foi positiva. Até onde chega o nosso conhecimento, os resultados indicam, pela primeira vez, a presença de DNA do M. leprae na urina de pacientes com hanseníase. Outro fato importante obtido através do exame é que entre os pacientes tratados 66.6% (4/6 eram positivos enquanto entre os não tratados somente 20% (2/10 foram positivos. Pelos presentes dados indicativos parece que o tratamento melhora os resultados do

  4. Vertebrate DNA in fecal samples from bonobos and gorillas: evidence for meat consumption or artefact?

    Directory of Open Access Journals (Sweden)

    Michael Hofreiter

    Full Text Available BACKGROUND: Deciphering the behavioral repertoire of great apes is a challenge for several reasons. First, due to their elusive behavior in dense forest environments, great ape populations are often difficult to observe. Second, members of the genus Pan are known to display a great variety in their behavioral repertoire; thus, observations from one population are not necessarily representative for other populations. For example, bonobos (Pan paniscus are generally believed to consume almost no vertebrate prey. However, recent observations show that at least some bonobo populations may consume vertebrate prey more commonly than previously believed. We investigated the extent of their meat consumption using PCR amplification of vertebrate mitochondrial DNA (mtDNA segments from DNA extracted from bonobo feces. As a control we also attempted PCR amplifications from gorilla feces, a species assumed to be strictly herbivorous. PRINCIPAL FINDINGS: We found evidence for consumption of a variety of mammalian species in about 16% of the samples investigated. Moreover, 40% of the positive DNA amplifications originated from arboreal monkeys. However, we also found duiker and monkey mtDNA in the gorilla feces, albeit in somewhat lower percentages. Notably, the DNA sequences isolated from the two ape species fit best to the species living in the respective regions. This result suggests that the sequences are of regional origin and do not represent laboratory contaminants. CONCLUSIONS: Our results allow at least three possible and mutually not exclusive conclusions. First, all results may represent contamination of the feces by vertebrate DNA from the local environment. Thus, studies investigating a species' diet from feces DNA may be unreliable due to the low copy number of DNA originating from diet items. Second, there is some inherent difference between the bonobo and gorilla feces, with only the later ones being contaminated. Third, similar to bonobos, for

  5. Vertebrate DNA in fecal samples from bonobos and gorillas: evidence for meat consumption or artefact?

    Science.gov (United States)

    Hofreiter, Michael; Kreuz, Eva; Eriksson, Jonas; Schubert, Grit; Hohmann, Gottfried

    2010-02-25

    Deciphering the behavioral repertoire of great apes is a challenge for several reasons. First, due to their elusive behavior in dense forest environments, great ape populations are often difficult to observe. Second, members of the genus Pan are known to display a great variety in their behavioral repertoire; thus, observations from one population are not necessarily representative for other populations. For example, bonobos (Pan paniscus) are generally believed to consume almost no vertebrate prey. However, recent observations show that at least some bonobo populations may consume vertebrate prey more commonly than previously believed. We investigated the extent of their meat consumption using PCR amplification of vertebrate mitochondrial DNA (mtDNA) segments from DNA extracted from bonobo feces. As a control we also attempted PCR amplifications from gorilla feces, a species assumed to be strictly herbivorous. We found evidence for consumption of a variety of mammalian species in about 16% of the samples investigated. Moreover, 40% of the positive DNA amplifications originated from arboreal monkeys. However, we also found duiker and monkey mtDNA in the gorilla feces, albeit in somewhat lower percentages. Notably, the DNA sequences isolated from the two ape species fit best to the species living in the respective regions. This result suggests that the sequences are of regional origin and do not represent laboratory contaminants. Our results allow at least three possible and mutually not exclusive conclusions. First, all results may represent contamination of the feces by vertebrate DNA from the local environment. Thus, studies investigating a species' diet from feces DNA may be unreliable due to the low copy number of DNA originating from diet items. Second, there is some inherent difference between the bonobo and gorilla feces, with only the later ones being contaminated. Third, similar to bonobos, for which the consumption of monkeys has only recently been

  6. Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

    Science.gov (United States)

    Luo, Chengwei; Tsementzi, Despina; Kyrpides, Nikos; Read, Timothy; Konstantinidis, Konstantinos T

    2012-01-01

    Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

  7. Compatibility of DNA IQ™, QIAamp® DNA Investigator, and QIAsymphony® DNA Investigator® with various fingerprint treatments.

    Science.gov (United States)

    Lin, Sze-Wah; Ip, Stephen C Y; Lam, Tze-Tsun; Tan, Tung-Fai; Yeung, Wai-Lung; Tam, Wai-Ming

    2017-03-01

    Latent fingerprint and touch DNA are the two most important contact evidence for individualization in forensic science which provide complementary information that can lead to direct and unequivocal identification of the culprit. In order to retrieve useful information from both fingerprints and DNA, which are usually mingled together, one strategy is to perform fingerprint examination prior to DNA analysis since common DNA sampling technique such as swabbing could disturb or even destroy fingerprint details. Here, we describe the compatibility of three automatic DNA extraction systems, namely, DNA IQ™, QIAamp ® DNA Investigator, and QIAsymphony ® DNA Investigator ® , with respective to the effects of various fingerprint detection techniques. Our results demonstrate that Super Glue fingerprint treatment followed by DNA IQ™ extraction shows better effectiveness in DNA profiling. Aluminum powder dusting offers the least interference to the three DNA extraction systems above. Magnetic powder dusting, on the other hand, strongly impedes DNA recovery. Physical Developer is the most intrusive, which yields profiles with poor quality, including lower peak heights, poor peak height ratios, and poor intra-color balance. In terms of the choice of extraction method, DNA IQ™ system is recommended for sampling after fingerprint treatments, but not the two DNA Investigator systems.

  8. DNA repair deficiency in lymphocytes from patients with actinic keratosis

    International Nuclear Information System (INIS)

    Abo-Darub, J.M.; Mackie, R.; Pitts, J.D.

    1978-01-01

    DNA repair activity was measured in peripheral blood lymphocytes from 18 patients with Actinic Keratosis and 18 age-matched control subjects, by comparing the incorporation of 3 H-thymidine into cells after irradiation with ultraviolet light with that into unirradiated cells. The incorporation was followed autoradiographically or by measuring acid insoluble radioactivity in cells labelled in the presence of hydroxyurea. The repair activity in lymphocytes from Actinic keratosis patients was only 47.1% (+-6.5%) of that in cells from the control subjects

  9. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    Energy Technology Data Exchange (ETDEWEB)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R., E-mail: antero@cdtn.br [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia; Melo, Maria N., E-mail: melo@icb.ufmg.br [Departamento de Parasitologia. Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais, Belo Horizonte, MG (Brazil)

    2011-07-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with {sup 32}P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  10. Diagnosis of canine visceral leishmaniasis with radiolabelled probes: comparison of the kDNA PCR-hybridization with three molecular methods in different clinical samples

    International Nuclear Information System (INIS)

    Ferreira, Aline Leandra C.; Ferreira, Sidney A.; Carregal, Virginia M.; Andrade, Antero Silva R.

    2011-01-01

    Leishmania (Leishmania) chagasi is responsible for visceral leishmaniasis (VL) in Brazil and the dog is the main domestic reservoir. Disease control is based on the elimination of infected animals and the use of a sensitive and specific diagnostic test is necessary. The Brazilian VL control program emphasizes serologic surveys, mainly using the enzyme-linked immunosorbent assay (ELISA) and the immunofluorescence antibody test (IFAT), followed by the elimination of the seropositive dogs. However, these techniques present limitations in terms of sensitivity and specificity. The Polymerase Chain Reaction (PCR) associated to hybridization with DNA probes labeled with 32 P has been recognized as a valuable tool for Leishmania identification. In this study, the sensitivity of kDNA PCR hybridization method was compared with three other molecular methods: Internal Transcribed Spacer 1 Nested PCR (ITS-1nPCR), Leishmania nested PCR (LnPCR) and Seminested kDNA PCR (kDNA snPCR). The comparison was performed in different clinical specimens: conjunctival swab, skin, blood and bone marrow. A group of thirty symptomatic dogs, positive in the parasitological and serological tests, was used. When. The techniques targeting kDNA mini-circles (kDNA snPCR and KDNA PCR-hybridization) showed the worst result for blood samples. The KDNA-PCR hybridization showed the best sensitivity for conjunctival swab. By comparing the samples on the basis of positivity obtained by the sum of all methods, the blood showed the worst outcome (71/120).The bone marrow showed the highest positivity (106/120), followed by conjunctival swab (100/120) and skin (89/120). Since the bone marrow samples are unsuitable for routine epidemiological surveys, the conjunctival swab was recommended because it allows high sensitivity, especially when associated with kDNA PCR hybridization method, and is a noninvasive sampling method. (author)

  11. Direct Quantification of Campylobacter jejuni in Chicken Fecal Samples Using Real-Time PCR: Evaluation of Six Rapid DNA Extraction Methods

    DEFF Research Database (Denmark)

    Garcia Clavero, Ana Belén; Kamara, Judy N.; Vigre, Håkan

    2013-01-01

    of this study, the Easy-DNA (Invitrogen) method generated lower Ct values, the best amplification efficiency (AE = 93.2 %) and good precision (R squared = 0.996). The method NucleoSpin® Tissue was able to detect samples spiked with the lowest Campylobacter concentration level (10 CFU/ml) but the amplification...... efficiency was not optimal (AE = 139.5 %). DNA extraction methods Easy-DNA Invitrogen, MiniMAG® and NucleoSpin® Tissue produced good real-time PCR reproducibility generating standard deviations from 0.3 to 0.8 between replicates....

  12. Dangers resulting from DNA profiling of biological materials derived from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT with regard to forensic genetic analysis

    Directory of Open Access Journals (Sweden)

    Renata Jacewicz

    2016-07-01

    Full Text Available The study documents the risk that comes with DNA analysis of materials derived from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT in forensic genetics. DNA chimerism was studied in 30 patients after allo-HSCT, based on techniques applied in contemporary forensic genetics, i.e. real-time PCR and multiplex PCR-STR with the use of autosomal DNA as well as Y-DNA markers. The results revealed that the DNA profile of the recipient’s blood was identical with the donor’s in the majority of cases. Therefore, blood analysis can lead to false conclusions in personal identification as well as kinship analysis. An investigation of buccal swabs revealed a mixture of DNA in the majority of recipients. Consequently, personal identification on the basis of stain analysis of the same origin may be impossible. The safest (but not ideal material turned out to be the hair root. Its analysis based on autosomal DNA revealed 100% of the recipient’s profile. However, an analysis based on Y-chromosome markers performed in female allo-HSCT recipients with male donors demonstrated the presence of donor DNA in hair cells – similarly to the blood and buccal swabs. In the light of potential risks arising from DNA profiling of biological materials derived from persons after allotransplantation in judicial aspects, certain procedures were proposed to eliminate such dangers. The basic procedures include abandoning the approach based exclusively on blood collection, both for kinship analysis and personal identification; asking persons who are to be tested about their history of allo-HSCT before sample collection and profile entry in the DNA database, and verification of DNA profiling based on hair follicles in uncertain cases.

  13. Radioresistant DNA synthesis in cells of patients showing increased chromosomal sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Barenfeld, L.S.; Pleskach, N.M.; Bildin, V.N.; Prokofjeva, V.V.; Mikhelson, V.M.

    1986-01-01

    The rate of DNA synthesis after γ-irradiation was studied either by analysis of the steady-state distribution of daughter [ 3 H]DNA in alkaline sucrose gradients or by direct assay of the amount of [ 3 H]thymidine incorporated into DNA of fibroblasts derived from a normal donor (LCH882) and from Down's syndrome (LCH944), Werner's syndrome (WS1LE) and xeroderma pigmentosum (XP2LE) patients with chromosomal sensitivity to ionizing radiation. Doses of γ-irradiation that markedly inhibited the rate of DNA synthesis in normal human cells caused almost no inhibition of DNA synthesis in the cells from the affected individuals. The radioresistant DNA synthesis in Down's syndrome cells was mainly due to a much lower inhibition of replicon initiation than that in normal cells; these cells were also more resistant to damage that inhibited replicon elongation. Our data suggest that radioresistant DNA synthesis may be an intrinsic feature of all genetic disorders showing increased radiosensitivity in terms of chromosome aberrations. (orig.)

  14. Fragmentation of sperm DNA using the TUNEL method.

    Science.gov (United States)

    Chenlo, P H; Curi, S M; Pugliese, M N; Ariagno, J I; Sardi-Segovia, M; Furlan, M J; Repetto, H E; Zeitler, E; Cohen, M; Mendeluk, G R

    2014-11-01

    To establish the validity of the TUNEL assay in determining sperm DNA fragmentation, the relationship between the degree of fragmentation and the seminal parameters and the sample needed to conduct the test. We used semen samples from healthy fertile men (n=33), patients who consulted for infertility with a prescription for the TUNEL assay (n=77) and patients with intracytoplasmic sperm injection failure (n=20), analyzed according to the 2010 WHO. The TUNEL/propidium iodide test was performed by flow cytometry, on baseline and post-swim-up samples. The cutoff value for the TUNEL assay (ROC curves) was 26%, with a sensitivity and specificity of 85% and 89%, respectively. The pre-swim-up and post-swim-up medians of the results from the TUNEL assay showed no significant differences (17.0% vs. 12.9%, respectively). However, 39.1% of the samples showed a difference greater than 15 in absolute value between the results of the baseline and post-swim-up TUNEL assays. The linear correlation study of the morphology, mobility and vitality using the post-swim-up TUNEL assay showed a greater correlation than preselection, with significant results (r: -0.394, P<.0001; r: -0.461, P<.0001; r: -0.526, P<.0001). The TUNEL assay is a valid test for clinical use. DNA fragmentation is a factor independent from traditional semen tests. We found a greater susceptibility to damage generated in the laboratory procedures in the samples with lower quality. The sample of choice for evaluating DNA fragmentation will depend on whether the clinician is treating a natural or assisted fertilization. Copyright © 2014 AEU. Published by Elsevier Espana. All rights reserved.

  15. Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients

    DEFF Research Database (Denmark)

    Poulsen, Nicklas N; Pedersen, Morten E; Østergaard, Jesper

    2016-01-01

    Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity......, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity....

  16. Modification of gDNA extraction from soil for PCR designed for the routine examination of soil samples contaminated with Toxocara spp. eggs.

    Science.gov (United States)

    Borecka, A; Gawor, J

    2008-06-01

    A modification of gDNA extraction was developed for the polymerase chain reaction (PCR) technique, intended for the detection and differentiation of Toxocara spp. eggs in soil or sediments. Sand samples from sandpits confirmed as being contaminated with Toxocara spp. eggs by the flotation technique were analysed by PCR. The use of proteinase K made it possible to obtain genomic DNA from the sample without needing to isolate eggs using flotation or to inactivate PCR inhibitors present in the sand. Specific primers in the PCR reaction allowed discrimination between T. canis and T. cati eggs. The modification simplified the procedure, thanks to eliminating the step of gDNA isolation from eggs, which is both laborious and difficult.

  17. Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

    Science.gov (United States)

    Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

    2016-03-01

    Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

  18. Identification of multiple mRNA and DNA sequences from small tissue samples isolated by laser-assisted microdissection.

    Science.gov (United States)

    Bernsen, M R; Dijkman, H B; de Vries, E; Figdor, C G; Ruiter, D J; Adema, G J; van Muijen, G N

    1998-10-01

    Molecular analysis of small tissue samples has become increasingly important in biomedical studies. Using a laser dissection microscope and modified nucleic acid isolation protocols, we demonstrate that multiple mRNA as well as DNA sequences can be identified from a single-cell sample. In addition, we show that the specificity of procurement of tissue samples is not compromised by smear contamination resulting from scraping of the microtome knife during sectioning of lesions. The procedures described herein thus allow for efficient RT-PCR or PCR analysis of multiple nucleic acid sequences from small tissue samples obtained by laser-assisted microdissection.

  19. Analysis of colorectal cancer and polyp for presence herpes simplex virus and cytomegalovirus DNA sequences by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Sahar Mehrabani khasraghi

    2016-05-01

    Full Text Available Introduction: In recent years, it was demonstrated that there is a clear association between the complicated course of colorectal cancer (CRC and the presence of herpes viruses. Despite a great number of published reports, the exact pathogenic role of herpes viruses remains unclear in these patients. The purpose of this study is to explore the prevalence of herpes simplex virus (HSV and cytomegalovirus (CMV in patients with CRC and polyp in comparison with healthy subjects using the polymerase chain reaction (PCR method. Methods: In this case-control study, 15 biopsies of patients with CRC and 20 colorectal polyp sample were selected. From each patient, two tissue samples were obtained: one sample from malignant tissue, and the other from normal colorectal tissue in an area located 15 cm away from the malignant tissue. Furthermore, 35 samples from healthy people as controls were selected. After DNA extraction, PCR was used to determine HSV and CMV genomes by specific primers. A statistical analysis was performed using the chi-square test. Results: Five CRC patients (33.3% had HSV DNA detected in both the malignant and the matched normal tissue. Five CRC patients (33.3% and seven polyp patients (35.0% had CMV DNA detected in both the malignant and the matched normal tissue. HSV DNA was found in 20% and CMV DNA in 37.1% of samples from healthy people as a control group. Thus, no significant association was observed between the prevalence of HSV and CMV, and an incidence of CRC and polyps according to the location of the samples as compared with the control group. Conclusion: The findings demonstrated that there is no direct molecular evidence to support the association between HSV and CMV and human colorectal malignancies. However, the results from this study do not exclude a possible oncogenic role of these viruses in the neoplastic development of colon cells.

  20. enDNA-Prot: Identification of DNA-Binding Proteins by Applying Ensemble Learning

    Directory of Open Access Journals (Sweden)

    Ruifeng Xu

    2014-01-01

    Full Text Available DNA-binding proteins are crucial for various cellular processes, such as recognition of specific nucleotide, regulation of transcription, and regulation of gene expression. Developing an effective model for identifying DNA-binding proteins is an urgent research problem. Up to now, many methods have been proposed, but most of them focus on only one classifier and cannot make full use of the large number of negative samples to improve predicting performance. This study proposed a predictor called enDNA-Prot for DNA-binding protein identification by employing the ensemble learning technique. Experiential results showed that enDNA-Prot was comparable with DNA-Prot and outperformed DNAbinder and iDNA-Prot with performance improvement in the range of 3.97–9.52% in ACC and 0.08–0.19 in MCC. Furthermore, when the benchmark dataset was expanded with negative samples, the performance of enDNA-Prot outperformed the three existing methods by 2.83–16.63% in terms of ACC and 0.02–0.16 in terms of MCC. It indicated that enDNA-Prot is an effective method for DNA-binding protein identification and expanding training dataset with negative samples can improve its performance. For the convenience of the vast majority of experimental scientists, we developed a user-friendly web-server for enDNA-Prot which is freely accessible to the public.

  1. Analysis of relationship between tumor markers and quantification of free DNA in serum of lung cancer patients

    International Nuclear Information System (INIS)

    Yang Shunfang; Zhang Peiling; Cao Jie; Zeng Jun; Dong Qianggang

    2006-01-01

    To evaluate the diagnostic value and relationship between five tumor markers (CA19- 9,CA125,CYFRA21-1 ,CEA,NSE) and free DNA in serum for lung cancer detection and try to find a new and more efficient tumor marker, the amounts of CA19-9, CA125, CYFRA21-1, CEA, NSE were determined by RIA and free DNA was determined by the use of quantitative real time PCR amplification of the human epidermal growth factor receptor (EGFR) in 52 lung cancer patients and 8 cases of benign pulmonary disease and 10 healthy controls. The resulls showed that average concentration of free DNA in serum of lung cancer patients, benign pulmo- nary disease and healthy controls was 107.6ng/mL, 76.86ng/mL and 18.8ng/mL, respective- ly. The diagnostic sensitivity, specificity and accuracy of free DNA for lung cancer were 71. 2%, 50% and 68.3%, same as the diagnostic value of combined detection of five tumor markers. The sensitivity, specificity and accuracy of the five tumor markers and free DNA combinend detection for lung cancer were 94.2%, 25% and 85%, respectively. The free DNA in the serum of lung cancer patients may be a new and better tumor marker. (authors)

  2. Test performance and acceptability of self- versus provider-collected swabs for high-risk HPV DNA testing in female-to-male trans masculine patients.

    Science.gov (United States)

    Reisner, Sari L; Deutsch, Madeline B; Peitzmeier, Sarah M; White Hughto, Jaclyn M; Cavanaugh, Timothy P; Pardee, Dana J; McLean, Sarah A; Panther, Lori A; Gelman, Marcy; Mimiaga, Matthew J; Potter, Jennifer E

    2018-01-01

    High-risk human papillomavirus (hrHPV) causes virtually all cervical cancers. Trans masculine (TM) people (those assigned female at birth who identify with a gender other than female) have low uptake of conventional cervical cancer screening. Self-collected hrHPV DNA testing has high levels of acceptability among cisgender (non-transgender) females and may support increased cervical cancer screening uptake in TM individuals. To assess the test performance and acceptability of self-collected vaginal specimens in comparison to provider-collected cervical swabs for hrHPV DNA detection in TM individuals ages 21-64 years. Between March 2015-September 2016, 150 TM participants with a cervix (mean age = 27.5 years; SD = 5.7) completed a one-time study visit comprised of a self-report survey, self-collected vaginal HPV DNA swab, clinician-administered cervical HPV swab, and brief interview on acceptability of clinical procedures. Participants were randomized to complete either self- or provider-collection first to minimize ordering effects. Self- and provider-collected samples were tested for 13 hrHPV DNA types using a DNA Hybridization Assay. The primary outcome variable was the concordance (kappa statistic) and performance (sensitivity, specificity) of self-collected vaginal HPV DNA specimens versus provider-collected cervical HPV swabs as the gold standard. Of the 131 participants completing both the self- and provider-collected HPV tests, 21 cases of hrHPV were detected by the provider cervical swab (gold standard; 16.0% hrHPV prevalence); 15 of these cases were accurately detected by the self-collected vaginal swab (71.4% concordance) (Kappa = 0.75, 95% Confidence Interval [CI]: 0.59, 0.92; p<0.001). Compared to the provider-collected cervical hrHPV DNA sample (gold standard), the self-collected vaginal hrHPV DNA test demonstrated a sensitivity of 71.4% (95% CI: 0.52, 0.91; p = 0.0495) and specificity of 98.2% (95% CI: 0.96, 1.00; p<0.0001). Over 90% of participants

  3. DNA Fingerprinting of Single Colonies of Helicobacter pylori from Gastric Cancer Patients Suggests Infection with a Single Predominant Strain

    OpenAIRE

    Miehlke, Stephan; Thomas, Rachel; Guiterrez, Oscar; Graham, David Y.; Go, Mae F.

    1999-01-01

    In each of six gastric cancer patients, repetitive extragenic palindromic PCR DNA fingerprints of 18 single colonies of Helicobacter pylori from the gastric antrum, corpus, and cardia were identical and matched that of the parental isolate. In three additional gastric cancer patients, 17 of 18 single-colony DNA fingerprints were identical to each other and to the DNA fingerprint of the corresponding parental isolate.

  4. Performance of mitochondrial DNA mutations detecting early stage cancer

    International Nuclear Information System (INIS)

    Jakupciak, John P; Srivastava, Sudhir; Sidransky, David; O'Connell, Catherine D; Maragh, Samantha; Markowitz, Maura E; Greenberg, Alissa K; Hoque, Mohammad O; Maitra, Anirban; Barker, Peter E; Wagner, Paul D; Rom, William N

    2008-01-01

    Mutations in the mitochondrial genome (mtgenome) have been associated with cancer and many other disorders. These mutations can be point mutations or deletions, or admixtures (heteroplasmy). The detection of mtDNA mutations in body fluids using resequencing microarrays, which are more sensitive than other sequencing methods, could provide a strategy to measure mutation loads in remote anatomical sites. We determined the mtDNA mutation load in the entire mitochondrial genome of 26 individuals with different early stage cancers (lung, bladder, kidney) and 12 heavy smokers without cancer. MtDNA was sequenced from three matched specimens (blood, tumor and body fluid) from each cancer patient and two matched specimens (blood and sputum) from smokers without cancer. The inherited wildtype sequence in the blood was compared to the sequences present in the tumor and body fluid, detected using the Affymetrix Genechip ® Human Mitochondrial Resequencing Array 1.0 and supplemented by capillary sequencing for noncoding region. Using this high-throughput method, 75% of the tumors were found to contain mtDNA mutations, higher than in our previous studies, and 36% of the body fluids from these cancer patients contained mtDNA mutations. Most of the mutations detected were heteroplasmic. A statistically significantly higher heteroplasmy rate occurred in tumor specimens when compared to both body fluid of cancer patients and sputum of controls, and in patient blood compared to blood of controls. Only 2 of the 12 sputum specimens from heavy smokers without cancer (17%) contained mtDNA mutations. Although patient mutations were spread throughout the mtDNA genome in the lung, bladder and kidney series, a statistically significant elevation of tRNA and ND complex mutations was detected in tumors. Our findings indicate comprehensive mtDNA resequencing can be a high-throughput tool for detecting mutations in clinical samples with potential applications for cancer detection, but it is

  5. Sampling the genomic pool of protein tyrosine kinase genes using the polymerase chain reaction with genomic DNA.

    Science.gov (United States)

    Oates, A C; Wollberg, P; Achen, M G; Wilks, A F

    1998-08-28

    The polymerase chain reaction (PCR), with cDNA as template, has been widely used to identify members of protein families from many species. A major limitation of using cDNA in PCR is that detection of a family member is dependent on temporal and spatial patterns of gene expression. To circumvent this restriction, and in order to develop a technique that is broadly applicable we have tested the use of genomic DNA as PCR template to identify members of protein families in an expression-independent manner. This test involved amplification of DNA encoding protein tyrosine kinase (PTK) genes from the genomes of three animal species that are well known development models; namely, the mouse Mus musculus, the fruit fly Drosophila melanogaster, and the nematode worm Caenorhabditis elegans. Ten PTK genes were identified from the mouse, 13 from the fruit fly, and 13 from the nematode worm. Among these kinases were 13 members of the PTK family that had not been reported previously. Selected PTKs from this screen were shown to be expressed during development, demonstrating that the amplified fragments did not arise from pseudogenes. This approach will be useful for the identification of many novel members of gene families in organisms of agricultural, medical, developmental and evolutionary significance and for analysis of gene families from any species, or biological sample whose habitat precludes the isolation of mRNA. Furthermore, as a tool to hasten the discovery of members of gene families that are of particular interest, this method offers an opportunity to sample the genome for new members irrespective of their expression pattern.

  6. Clinical symptoms and DNA repair characteristics of xeroderma pigmentosum patients from Germany

    International Nuclear Information System (INIS)

    Thielmann, H.W.; Popanda, O.; Edler, L.; Jung, E.G.

    1991-01-01

    Sixty-one xeroderma pigmentosum (XP) patients living in the Federal Republic of Germany were investigated. Clinical symptoms were correlated with DNA repair parameters measured in fibroblasts grown from skin biopsies. Classification according to the international complementation groups revealed that of the 61 patients 3 belonged to group A, 26 to group C, 16 to group D, 3 to group E, and 2 to group F; 11 were of the XP variant type. A striking clinical aspect was the frequency of histogenetically different skin tumors varying from one XP complementation group to the other: squamous and basal cell carcinomas predominated in XP group C; lentigo maligna melanomas were most frequent in group D; basal cell carcinomas occurred preferentially in group E and XP variants. Three DNA repair parameters were determined for 46 fibroblast strains: colony-forming ability (D0); DNA repair synthesis (G0); and DNA-incising capacity (E0). Dose-response experiments with up to 13 dose levels were performed throughout to achieve sufficient experimental accuracy. DNA-damaging treatments included UV light, the 'UV-like' carcinogen N-acetoxy-2-acetylaminofluorene, and the alkylating carcinogens methyl methanesulfonate and N-methyl-N-nitrosourea. Comparison of clinical signs and repair data was made on the basis of D0, G0, and E0 values of both individual cell strains and weighted means of XP complementation groups. Despite considerable clinical and biochemical heterogeneity within complementation groups distinctive features emerged. In general, D0, G0, and E0 values of all XP strains investigated, including XP variants, were found to be reduced upon treatment with UV light or N-acetoxy-2-acetylaminofluorene

  7. Altered DNA Methylation and Expression Profiles of 8-Oxoguanine DNA Glycosylase 1 in Lens Tissue from Age-related Cataract Patients.

    Science.gov (United States)

    Wang, Yong; Li, Fei; Zhang, Guowei; Kang, Lihua; Qin, Bai; Guan, Huaijin

    2015-01-01

    Oxidative stress and DNA damage contribute to the pathogenesis of age-related cataract (ARC). Most oxidative DNA lesions are repaired via the base excision repair (BER) proteins including 8-oxoguanine DNA glycosylase 1 (OGG1). This study examined DNA methylation of CpG islands upstream of OGG1 and their relation to the gene expression in lens cortex from ARC patients. The clinical case-control study consisted of 15 cortical type of ARC patients and 15 age-matched non-ARC controls who received transparent lens extraction due to vitreoretinal diseases. OGG1 expression in lens cortex was analyzed by qRT-PCR and Western blot. The localization and the proportion of cells positive for OGG1 were determined by immunofluorescence. Bisulfite-sequencing PCR (BSP) was performed to evaluate the methylation status of CpG islands near OGG1 in DNA extracted from lens cortex. To test relationship between the methylation and the expression of the gene of interest, 5-Aza-2'-deoxycytidine (5-Aza-dC) was used to induce demethylation of cultured human lens epithelium B-3 (HLE B-3). To test the role of OGG1 in the repair of cellular damage, HLE B-3 was transfected with OGG1 vector, followed by ultraviolet radiation b (UVB) exposure to induce apoptosis. The mRNA and protein levels of OGG1 were significantly reduced in the lens cortex of ARC. Immunofluorescence showed that the proportion of OGG1-positive cells decreased significantly in ARC cortex in comparison with the control. The CpG island in first exon of OGG1 displayed hypermethylation in the DNA extracted from the lens cortex of ARC. Treatment of HLEB-3 cells with 5-Aza-dC upregulated OGG1 expression. UVB-induced apoptosis was attenuated after transfection with OGG1. A reduced OGG1 expression was correlated with hypermethylation of a CpG island of OGG1 in lens cortex of ARC. The role of epigenetic change in OGG1 gene in the susceptibility to oxidative stress induced cortical ARC is warranted to further study.

  8. N-acetylcysteine normalizes the urea cycle and DNA repair in cells from patients with Batten disease.

    Science.gov (United States)

    Kim, June-Bum; Lim, Nary; Kim, Sung-Jo; Heo, Tae-Hwe

    2012-12-01

    Batten disease is an inherited disorder characterized by early onset neurodegeneration due to the mutation of the CLN3 gene. The function of the CLN3 protein is not clear, but an association with oxidative stress has been proposed. Oxidative stress and DNA damage play critical roles in the pathogenesis of neurodegenerative diseases. Antioxidants are of interest because of their therapeutic potential for treating neurodegenerative diseases. We tested whether N-acetylcysteine (NAC), a well-known antioxidant, improves the pathology of cells from patients with Batten disease. At first, the expression levels of urea cycle components and DNA repair enzymes were compared between Batten disease cells and normal cells. We used both mRNA expression levels and Western blot analysis. We found that carbamoyl phosphate synthetase 1, an enzyme involved in the urea cycle, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta, enzymes involved in DNA repair, were expressed at higher levels in Batten disease cells than in normal cells. The treatment of Batten disease cells with NAC for 48 h attenuated activities of the urea cycle and of DNA repair, as indicated by the substantially decreased expression levels of carbamoyl phosphate synthetase 1, 8-oxoguanine DNA glycosylase 1 and DNA polymerase beta proteins compared with untreated Batten cells. NAC may serve in alleviating the burden of urea cycle and DNA repair processes in Batten disease cells. We propose that NAC may have beneficial effects in patients with Batten disease. Copyright © 2012 John Wiley & Sons, Ltd.

  9. Massively parallel DNA sequencing facilitates diagnosis of patients with Usher syndrome type 1.

    Directory of Open Access Journals (Sweden)

    Hidekane Yoshimura

    Full Text Available Usher syndrome is an autosomal recessive disorder manifesting hearing loss, retinitis pigmentosa and vestibular dysfunction, and having three clinical subtypes. Usher syndrome type 1 is the most severe subtype due to its profound hearing loss, lack of vestibular responses, and retinitis pigmentosa that appears in prepuberty. Six of the corresponding genes have been identified, making early diagnosis through DNA testing possible, with many immediate and several long-term advantages for patients and their families. However, the conventional genetic techniques, such as direct sequence analysis, are both time-consuming and expensive. Targeted exon sequencing of selected genes using the massively parallel DNA sequencing technology will potentially enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using this technique combined with direct sequence analysis, we screened 17 unrelated Usher syndrome type 1 patients and detected probable pathogenic variants in the 16 of them (94.1% who carried at least one mutation. Seven patients had the MYO7A mutation (41.2%, which is the most common type in Japanese. Most of the mutations were detected by only the massively parallel DNA sequencing. We report here four patients, who had probable pathogenic mutations in two different Usher syndrome type 1 genes, and one case of MYO7A/PCDH15 digenic inheritance. This is the first report of Usher syndrome mutation analysis using massively parallel DNA sequencing and the frequency of Usher syndrome type 1 genes in Japanese. Mutation screening using this technique has the power to quickly identify mutations of many causative genes while maintaining cost-benefit performance. In addition, the simultaneous mutation analysis of large numbers of genes is useful for detecting mutations in different genes that are possibly disease modifiers or of digenic inheritance.

  10. Massively parallel DNA sequencing facilitates diagnosis of patients with Usher syndrome type 1.

    Science.gov (United States)

    Yoshimura, Hidekane; Iwasaki, Satoshi; Nishio, Shin-Ya; Kumakawa, Kozo; Tono, Tetsuya; Kobayashi, Yumiko; Sato, Hiroaki; Nagai, Kyoko; Ishikawa, Kotaro; Ikezono, Tetsuo; Naito, Yasushi; Fukushima, Kunihiro; Oshikawa, Chie; Kimitsuki, Takashi; Nakanishi, Hiroshi; Usami, Shin-Ichi

    2014-01-01

    Usher syndrome is an autosomal recessive disorder manifesting hearing loss, retinitis pigmentosa and vestibular dysfunction, and having three clinical subtypes. Usher syndrome type 1 is the most severe subtype due to its profound hearing loss, lack of vestibular responses, and retinitis pigmentosa that appears in prepuberty. Six of the corresponding genes have been identified, making early diagnosis through DNA testing possible, with many immediate and several long-term advantages for patients and their families. However, the conventional genetic techniques, such as direct sequence analysis, are both time-consuming and expensive. Targeted exon sequencing of selected genes using the massively parallel DNA sequencing technology will potentially enable us to systematically tackle previously intractable monogenic disorders and improve molecular diagnosis. Using this technique combined with direct sequence analysis, we screened 17 unrelated Usher syndrome type 1 patients and detected probable pathogenic variants in the 16 of them (94.1%) who carried at least one mutation. Seven patients had the MYO7A mutation (41.2%), which is the most common type in Japanese. Most of the mutations were detected by only the massively parallel DNA sequencing. We report here four patients, who had probable pathogenic mutations in two different Usher syndrome type 1 genes, and one case of MYO7A/PCDH15 digenic inheritance. This is the first report of Usher syndrome mutation analysis using massively parallel DNA sequencing and the frequency of Usher syndrome type 1 genes in Japanese. Mutation screening using this technique has the power to quickly identify mutations of many causative genes while maintaining cost-benefit performance. In addition, the simultaneous mutation analysis of large numbers of genes is useful for detecting mutations in different genes that are possibly disease modifiers or of digenic inheritance.

  11. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

    Science.gov (United States)

    Matthaios, Dimitrios; Balgkouranidou, Ioanna; Karayiannakis, Anastasios; Bolanaki, Helen; Xenidis, Nikolaos; Amarantidis, Kyriakos; Chelis, Leonidas; Romanidis, Konstantinos; Chatzaki, Aikaterini; Lianidou, Evi; Trypsianis, Grigorios; Kakolyris, Stylianos

    2016-07-01

    DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli ( APC ) and Ras association domain family 1 isoform A ( RASSF1A ) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, PAPC . Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, PAPC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

  12. Analysis of hepatitis B virus intrahepatic covalently closed circular DNA and serum viral markers in treatment-naive patients with acute and chronic HBV infection.

    Directory of Open Access Journals (Sweden)

    Weijie Li

    Full Text Available BACKGROUND: This study aimed to investigate the relationships of intrahepatic cccDNA with serum HBsAg and with HBV DNA in treatment-naive patients throughout acute and chronic HBV infection. METHODS: A total of 120 patients who had a liver biopsy were enrolled, including 19 with acute hepatitis B (AHB, and 101 patients with chronic HBV infection (CHB of whom were 10 in immune-tolerant (IT phase, 59 in immune-clearance (IC phase, 8 in low-replicative (LR phase, and 24 in HBeAg-negative hepatitis (ENH phase. Intrahepatic cccDNA, serum HBsAg and serum HBV DNA levels were comparatively analyzed. RESULTS: The median intrahepatic cccDNA levels were 0.18 4.80, 3.81, 0.22 and 0.97 copies/cell for patients with AHB, CHB-IT, CHB-IC, CHB-LR, and CHB-ENH, respectively. In AHB patients, intrahepatic cccDNA was positively correlated with serum HBsAg (r = 0.665, P = 0.003, as well as serum HBV DNA (r = 0.536, P = 0.022. In CHB patients, intrahepatic cccDNA was positively correlated with serum HBsAg in the IC phase (r = 0.392, P = 0.005, and with serum HBV DNA in the IC phase (r = 0.301, P = 0.036 and ENH phase (r = 0.588, P = 0.013. HBV replicative efficiency, defined as the ratio of serum HBV DNA to intrahepatic cccDNA, was obviously lower in AHB and CHB-LR patients than in CHB-IT, CHB-IC and CHB-ENH patients (0.70 and 0.53 vs. 1.12, 1.09 and 0.99, P<0.001, values were logarithmic transformed for analysis. In CHB-IC patients, HBV replicative efficiency was positively correlated with histological activity index of liver inflammation (r = 0.308, P = 0.009. CONCLUSION: Serum HBsAg and HBV DNA levels may reflect the amount of active intrahepatic cccDNA in treatment-naive AHB and CHB-IC patients. Reduced intrahepatic cccDNA and HBV replicative efficiency may imply effective immune control of HBV infection.

  13. New polymorphisms of Xeroderma Pigmentosum DNA repair genes in myelodysplastic syndrome.

    Science.gov (United States)

    Santiago, Sabrina Pinheiro; Junior, Howard Lopes Ribeiro; de Sousa, Juliana Cordeiro; de Paula Borges, Daniela; de Oliveira, Roberta Taiane Germano; Farias, Izabelle Rocha; Costa, Marília Braga; Maia, Allan Rodrigo Soares; da Nóbrega Ito, Mayumi; Magalhães, Silvia Maria Meira; Pinheiro, Ronald Feitosa

    2017-07-01

    The association between Xeroderma Pigmentosum DNA repair genes (XPA rs1800975, XPC rs2228000, XPD rs1799793 and XPF rs1800067) polymorphisms and myelodysplastic syndrome (MDS) have not been reported. To assess the functional role between these polymorphisms and MDS, we evaluated 189 samples stratified in two groups: 95 bone marrow samples from MDS patients and 94 from healthy elderly volunteers used as controls. Genotypes for all polymorphisms were identified in DNA samples in an allelic discrimination experiment by real-time polymerase chain reaction (qPCR). We also studied the mRNA expression of XPA and XPC genes to evaluate if its polymorphisms were functional in 53 RNAm MDS patients by qPCR methodologies. To the rs2228000 polymorphism, the CT and TT polymorphic genotype were associated with increased odds ratio (OR) of more profound cytopenia (hemoglobin and neutrophils count). To the rs1799793 polymorphism, we found that the GG homozygous wild-type genotype was associated with a decreased chance of developing MDS. We observed low expression of XPA in younger patients, in hypoplastic MDS and patients with abnormal karyotype when presented AG or AA polymorphic genotypes. We also found that there was a statistically significant interaction between the presence of micromegakaryocyte on down regulation of XPC regarding the CT heterozygous genotype of the rs1800975 polymorphism. Our results suggest that new functional polymorphisms of Xeroderma Pigmentosum DNA repair genes in MDS are related to its pathogenesis and prognosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

    Science.gov (United States)

    Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schröder, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinhäusel, Andreas; Egger, Gerda

    2015-01-01

    Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. Results: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. Conclusions: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer. PMID:26335606

  15. Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2012-01-01

    Full Text Available Abstract Background The central role of the somatotrophic axis in animal post-natal growth, development and fertility is well established. Therefore, the identification of genetic variants affecting quantitative traits within this axis is an attractive goal. However, large sample numbers are a pre-requisite for the identification of genetic variants underlying complex traits and although technologies are improving rapidly, high-throughput sequencing of large numbers of complete individual genomes remains prohibitively expensive. Therefore using a pooled DNA approach coupled with target enrichment and high-throughput sequencing, the aim of this study was to identify polymorphisms and estimate allele frequency differences across 83 candidate genes of the somatotrophic axis, in 150 Holstein-Friesian dairy bulls divided into two groups divergent for genetic merit for fertility. Results In total, 4,135 SNPs and 893 indels were identified during the resequencing of the 83 candidate genes. Nineteen percent (n = 952 of variants were located within 5' and 3' UTRs. Seventy-two percent (n = 3,612 were intronic and 9% (n = 464 were exonic, including 65 indels and 236 SNPs resulting in non-synonymous substitutions (NSS. Significant (P ® MassARRAY. No significant differences (P > 0.1 were observed between the two methods for any of the 43 SNPs across both pools (i.e., 86 tests in total. Conclusions The results of the current study support previous findings of the use of DNA sample pooling and high-throughput sequencing as a viable strategy for polymorphism discovery and allele frequency estimation. Using this approach we have characterised the genetic variation within genes of the somatotrophic axis and related pathways, central to mammalian post-natal growth and development and subsequent lactogenesis and fertility. We have identified a large number of variants segregating at significantly different frequencies between cattle groups divergent for calving

  16. Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

    Directory of Open Access Journals (Sweden)

    Chengwei Luo

    Full Text Available Next-generation sequencing (NGS is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage correlated highly between the two platforms (R(2>0.9. Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

  17. Incidence of human papilloma virus in esophageal squamous cell carcinoma in patients from the Lublin region.

    Science.gov (United States)

    Dąbrowski, Andrzej; Kwaśniewski, Wojciech; Skoczylas, Tomasz; Bednarek, Wiesława; Kuźma, Dorota; Goździcka-Józefiak, Anna

    2012-10-28

    To assess the prevalence of human papilloma virus (HPV) in esophageal squamous cell carcinoma (ESCC) in the south-eastern region of Poland. The study population consisted of 56 ESCC patients and 35 controls. The controls were patients referred to our department due to other non-esophageal and non-oncological disorders with no gross or microscopic esophageal pathology as confirmed by endoscopy and histopathology. In the ESCC patients, samples were taken from normal mucosa (56 mucosa samples) and from the tumor (56 tumor samples). Tissue samples from the controls were taken from normal mucosa of the middle esophagus (35 control samples). Quantitative determination of DNA was carried out using a spectrophotometric method. Genomic DNA was isolated using the QIAamp DNA Midi Kit. HPV infection was identified following PCR amplification of the HPV gene sequence, using primers MY09 and MY11 complementary to the genome sequence of at least 33 types of HPV. The sequencing results were computationally analyzed using the basic local alignment search tool database. In tumor samples, HPV DNA was identified in 28 of 56 patients (50%). High risk HPV phenotypes (16 or/and 18) were found in 5 of 56 patients (8.9%), low risk in 19 of 56 patients (33.9%) and other types of HPV (37, 81, 97, CP6108) in 4 of 56 patients (7.1%). In mucosa samples, HPV DNA was isolated in 21 of 56 patients (37.5%). High risk HPV DNA was confirmed in 3 of 56 patients (5.3%), low risk HPV DNA in 12 of 56 patients (21.4%), and other types of HPV in 6 of 56 patients (10.7%). In control samples, HPV DNA was identified in 4 of 35 patients (11.4%) with no high risk HPV. The occurrence of HPV in ESCC patients was significantly higher than in the controls [28 of 56 (50%) vs 4 of 35 (11.4%), P prevalence of HPV was identified in female patients (71.4% vs 46.9%). Accordingly, the high risk phenotypes were isolated more frequently in female patients and this difference reached statistical significance [3 of 7 (42

  18. Increased Plasma Cell-Free DNA Level during HTNV Infection: Correlation with Disease Severity and Virus Load

    Directory of Open Access Journals (Sweden)

    Jing Yi

    2014-07-01

    Full Text Available Cell-free DNA (cf-DNA in blood represents a promising DNA damage response triggered by virus infection or trauma, tumor, etc. Hantavirus primarily causes two diseases: haemorrhagic fever with renal syndrome (HFRS and Hantavirus cardiopulmonary syndrome (HCPS, depending on different Hantavirus species. The aim of this study was to evaluate plasma cf-DNA levels in acute phase of HFRS, and to correlate plasma cf-DNA with disease severity and plasma Hanttan virus (HTNV load. We observed the appearance of cf-DNA in 166 plasma samples from 76 HFRS patients: the plasma cf-DNA levels peaked at the hypotensive stage of HFRS, and then decreased gradually. Until the diuretic stage, there was no significant difference in plasma cf-DNA level between patients and the healthy control. Exclusively in the febrile/hypotensive stage, the plasma cf-DNA levels of severe/critical patients were higher than those of the mild/moderate group. Moreover, the plasma cf-DNA value in the early stage of HFRS was correlated with HTNV load and disease severity. In most of the patients, plasma cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA. In conclusion, the plasma cf-DNA levels were dynamically elevated during HFRS, and correlated with disease severity, which suggests that plasma cf-DNA may be a potential biomarker for the pathogenesis and prognosis of HFRS.

  19. Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

    International Nuclear Information System (INIS)

    Schneider, Katja U; Liebenberg, Volker; Kneip, Christoph; Seegebarth, Anke; Erdogan, Fikret; Rappold, Gudrun; Schmidt, Bernd; Dietrich, Dimo; Fleischhacker, Michael; Leschber, Gunda; Merk, Johannes; Schäper, Frank; Stapert, Henk R; Vossenaar, Erik R; Weickmann, Sabine

    2011-01-01

    DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples. SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH. A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference. Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples

  20. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    Science.gov (United States)

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  1. A nuclear DNA-based species determination and DNA quantification assay for common poultry species.

    Science.gov (United States)

    Ng, J; Satkoski, J; Premasuthan, A; Kanthaswamy, S

    2014-12-01

    DNA testing for food authentication and quality control requires sensitive species-specific quantification of nuclear DNA from complex and unknown biological sources. We have developed a multiplex assay based on TaqMan® real-time quantitative PCR (qPCR) for species-specific detection and quantification of chicken (Gallus gallus), duck (Anas platyrhynchos), and turkey (Meleagris gallopavo) nuclear DNA. The multiplex assay is able to accurately detect very low quantities of species-specific DNA from single or multispecies sample mixtures; its minimum effective quantification range is 5 to 50 pg of starting DNA material. In addition to its use in food fraudulence cases, we have validated the assay using simulated forensic sample conditions to demonstrate its utility in forensic investigations. Despite treatment with potent inhibitors such as hematin and humic acid, and degradation of template DNA by DNase, the assay was still able to robustly detect and quantify DNA from each of the three poultry species in mixed samples. The efficient species determination and accurate DNA quantification will help reduce fraudulent food labeling and facilitate downstream DNA analysis for genetic identification and traceability.

  2. A pilot study on the use of cerebrospinal fluid cell-free DNA in intramedullary spinal ependymoma.

    Science.gov (United States)

    Connolly, Ian David; Li, Yingmei; Pan, Wenying; Johnson, Eli; You, Linya; Vogel, Hannes; Ratliff, John; Hayden Gephart, Melanie

    2017-10-01

    Cerebrospinal fluid (CSF) represents a promising source of cell-free DNA (cfDNA) for tumors of the central nervous system. A CSF-based liquid biopsy may obviate the need for riskier tissue biopsies and serve as a means for monitoring tumor recurrence or response to therapy. Spinal ependymomas most commonly occur in adults, and aggressive resection must be delicately balanced with the risk of injury to adjacent normal tissue. In patients with subtotal resection, recurrence commonly occurs. A CSF-based liquid biopsy matched to the patient's spinal ependymoma mutation profile has potential to be more sensitive then surveillance MRI, but the utility has not been well characterized for tumors of the spinal cord. In this study, we collected matched blood, tumor, and CSF samples from three adult patients with WHO grade II intramedullary spinal ependymoma. We performed whole exome sequencing on matched tumor and normal DNA to design Droplet Digital™ PCR (ddPCR) probes for tumor and wild-type mutations. We then interrogated CSF samples for tumor-derived cfDNA by performing ddPCR on extracted cfDNA. Tumor cfDNA was not reliably detected in the CSF of our cohort. Anatomic sequestration and low grade of intramedullary spinal cord tumors likely limits the role of CSF liquid biopsy.

  3. Circulating tumor DNA functions as an alternative for tissue to overcome tumor heterogeneity in advanced gastric cancer.

    Science.gov (United States)

    Gao, Jing; Wang, Haixing; Zang, Wanchun; Li, Beifang; Rao, Guanhua; Li, Lei; Yu, Yang; Li, Zhongwu; Dong, Bin; Lu, Zhihao; Jiang, Zhi; Shen, Lin

    2017-09-01

    Overcoming tumor heterogeneity is a major challenge for personalized treatment of gastric cancer, especially for human epidermal growth factor receptor-2 targeted therapy. Analysis of circulating tumor DNA allows a more comprehensive analysis of tumor heterogeneity than traditional biopsies in lung cancer and breast cancer, but little is known in gastric cancer. We assessed mutation profiles of ctDNA and primary tumors from 30 patients with advanced gastric cancer, then performed a comprehensive analysis of tumor mutations by multiple biopsies from five patients, and finally analyzed the concordance of HER2 amplification in ctDNA and paired tumor tissues in 70 patients. By comparing with a single tumor sample, ctDNA displayed a low concordance of mutation profile, only approximately 50% (138/275) somatic mutations were found in paired tissue samples, however, when compared with multiple biopsies, most DNA mutations in ctDNA were also shown in paired tumor tissues. ctDNA had a high concordance (91.4%, Kappa index = 0.784, P < 0.001) of HER2 amplification with tumor tissues, suggesting it might be an alternative for tissue. It implied that ctDNA-based assessment could partially overcome the tumor heterogeneity, and might serve as a potential surrogate for HER2 analysis in gastric cancer. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  4. High load of Merkel cell polyomavirus DNA detected in the normal skin of Japanese patients with Merkel cell carcinoma.

    Science.gov (United States)

    Hashida, Yumiko; Nakajima, Kimiko; Nakajima, Hideki; Shiga, Takeo; Tanaka, Moe; Murakami, Masanao; Matsuzaki, Shigenobu; Naganuma, Seiji; Kuroda, Naoki; Seki, Yasutaka; Katano, Harutaka; Sano, Shigetoshi; Daibata, Masanori

    2016-09-01

    Although Merkel cell polyomavirus (MCPyV) has the potential to cause Merkel cell carcinoma (MCC), it is also found in the normal skin of healthy individuals. However, the mechanism for transformation of MCPyV to an oncogenic form is unknown. To investigate the levels of MCPyV infection in the normal skin patients with MCC compared with those in a control cohort. We studied a total of six Japanese patients with cutaneous MCC. Sun-exposed and sun-unexposed skin swabs were obtained and analyzed for MCPyV loads using quantitative real-time polymerase chain reaction. At first, we found a patient with MCC carrying an extremely high load of MCPyV DNA in normal skin. This unique case prompted us to further explore the levels of MCPyV as skin microbiota in patients with MCC. We showed that MCPyV DNA levels were significantly higher in swabs obtained from normal skin samples of six patients with MCC compared with those from 30 age-matched healthy individuals and 19 patients with other cutaneous cancers. Whereas MCPyV strains obtained from the normal skin of patients with MCC had gene sequences without structural alterations, sequences of the tumor-derived strains showed truncating mutations or deletions. Although the number of patients with MCC studied was small, our findings suggest that MCC may occur with a background of high MCPyV load in the skin, and are expected to stimulate further studies on whether such skin virome levels could be one of predictive markers for the development of MCC. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Unsatisfactory exfoliative anal cytology samples, 15-year experience with histologic, cytologic, and molecular follow-up.

    Science.gov (United States)

    Khattab, Ruba; McMeekin, Emily; Taege, Alan J; Hekman, James M; Brainard, Jennifer A; Underwood, Dawn; Procop, Gary W; Sturgis, Charles D

    2018-02-01

    The incidence of anal carcinoma has risen in recent decades. Exfoliative cytology screening of selected high risk patients is performed in many centers. Unsatisfactory cytology results are frustrating to patients, clinicians, and laboratorians. The aim of this study is to ascertain outcomes of patients with non-diagnostic anal cytology. A retrospective review of anal cytology testing performed at the Cleveland Clinic between 01/01/2001 and 12/31/2015 was performed. All cases were received as liquid-based samples and processed as ThinPreps (Hologic, Marlborough, MA). Co-testing for HR-HPV DNA was performed using Hybrid Capture 2® (Qiagen, Germantown, MD) in the majority of patients. Of 1,276 ThinPrep anal cytology samples, 130 (10%) were deemed unsatisfactory. 77% of patients were HIV positive. 85% were males. Of the unsatisfactory cases, 116 (89%) were co-tested for HR-HPV DNA. Of those, 40 patients (34%) had a simultaneous positive HR-HPV DNA. Adequate follow up cytology within a one year and a two year period revealed that 18/130 (14%) and 26/130 (20%) of patients had ASC or SIL respectively. Histologic follow-up within one and two years showed 3 patients (2%) and 8 patients (6%) with HSIL or worse. High risk patients with unsatisfactory anal cytology are not "negative". At least one-third proved to be concomitantly HR-HPV DNA positive with one-fifth showing subsequent cytologic squamous abnormalities and with more than 5% being diagnosed with a high grade intraepithelial lesion within two years. Prompt repeat cytology and/or HR-HPV DNA is recommended for high risk patients with non-diagnostic cytology. © 2017 Wiley Periodicals, Inc.

  6. The Stool DNA Test is More Accurate than the Plasma Septin 9 Test in Detecting Colorectal Neoplasia

    Science.gov (United States)

    Ahlquist, David A.; Taylor, William R.; Mahoney, Douglas W.; Zou, Hongzhi; Domanico, Michael; Thibodeau, Stephen N.; Boardman, Lisa A.; Berger, Barry M.; Lidgard, Graham P.

    2014-01-01

    Background & Aims Several noninvasive tests have been developed for colorectal cancer (CRC) screening. We compared the sensitivities of a multi-marker test for stool DNA (sDNA) and a plasma test for methylated Septin 9 (SEPT9) in identifying patients with large adenomas or CRC. Methods We analyzed paired stool and plasma samples from 30 patients with CRC and 22 with large adenomas from Mayo Clinic archives. Stool (n=46) and plasma (n=49) samples from age- and sex-matched patients with normal colonoscopy results were used as controls. The sDNA test is an assay for methylated BMP3, NDRG4, vimentin, and TFPI2; mutant KRAS; the β-actin gene, and quantity of hemoglobin (by the porphyrin method). It was performed blindly at Exact Sciences (Madison WI); the test for SEPT9 was performed at ARUP Laboratories (Salt Lake City UT). Results were considered positive based on the manufacturer's specificity cutoff values of 90% and 89%, respectively. Results The sDNA test detected adenomas (median 2 cm, range 1–5 cm) with 82% sensitivity (95% confidence interval [CI], 60%–95%); SEPT9 had 14% sensitivity (95% CI, 3%–35%; P=.0001). The sDNA test identified patients with CRC with 87% sensitivity (95% CI, 69%–96%); SEPT9 had 60% sensitivity (95% CI, 41%–77%; P=.046). The sDNA test identified patients with stage I–III CRC with 91% sensitivity (95% CI, 71%–99%); SEPT9 had 50% sensitivity (95% CI, 28%–72%; P=.013); for stage IV CRC, sensitivity values were 75% (95% CI, 35%–97%) and 88% (95% CI, 47%–100%), respectively (P=.56). False-positive rates were 7% for the sDNA test and 27% for SEPT9. Conclusions Based on analyses of paired samples, the sDNA test detects non-metastatic CRC and large adenomas with significantly greater levels of sensitivity than the SEPT9 test. These findings might be used to modify approaches for CRC prevention and early detection. PMID:22019796

  7. Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer (ITS) region of peritrich ciliates in environmental samples

    Science.gov (United States)

    Su, Lei; Zhang, Qianqian; Gong, Jun

    2017-07-01

    Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifications of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.

  8. Clinical utility of circulating cell-free DNA in advanced colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Allan A Lima Pereira

    Full Text Available Circulating cell-free DNA (cfDNA isolated from the plasma of cancer patients (pts has been shown to reflect the genomic mutation profile of the tumor. However, physician and patient assessment of clinical utility of these assays in patients with metastatic colorectal cancer (mCRC has not been previously described.Patients were prospectively consented to a prospective genomic matching protocol (Assessment of Targeted Therapies Against Colorectal Cancer [ATTACC], with collection of blood for cfDNA extraction and sequencing of a 54-gene panel in a CLIA-certified lab. Formalin-fixed, paraffin-embedded (FFPE tissue from prior resections or biopsies underwent 50-gene sequencing. Results from both assays were returned to the treating physicians for patient care and clinical trial selection. Follow-up surveys of treating physicians and chart reviews assessed clinical utility.128 mCRC pts were enrolled between 6/2014 and 1/2015. Results were returned in median of 13 and 26 days for cfDNA and FFPE sequencing, respectively. With cfDNA sequencing, 78% (100/128 of samples had a detectable somatic genomic alteration. 50% of cfDNA cases had potentially actionable alterations, and 60% of these could be genomically matched to at least one clinical trial in our institution. 50% (15/30 of these pts enrolled onto an identified matched trial. Physicians reported that the cfDNA testing improved the quality of care they could provide in 73% of the cases, and that 89% of pts reported greater satisfaction with the efforts to personalize experimental therapeutic agents.cfDNA sequencing can provide timely information on potentially actionable mutations and amplifications, thereby facilitating clinical trial enrollment and improving the perceived quality of care.

  9. Identification of episomal human papillomavirus and other DNA viruses in cytological anal samples of HIV-uninfected men who have sex with men.

    Directory of Open Access Journals (Sweden)

    Maria Gabriella Donà

    Full Text Available To date, there have been only few studies that investigated integration of anal Human Papillomavirus (HPV. Most of them were conducted on HIV-infected individuals and mainly analyzed samples from high-grade lesions and invasive cancer. We aimed to investigate HPV physical status in HIV-negative men who have sex with men (MSM with a detectable anal HPV infection, irrespective of the presence of lesions. We also sought to explore the presence of other circular DNA viruses in the anal region. Study participants were attendees of an STI screening program, which were also screened for anal HPV infection and cytological abnormalities. HPV physical status was assessed using multiply-primed RCA. HPV16-positive samples were also analyzed using E2/E6 multiplex PCR, qRT-PCR and APOT assay. RCA and virus-specific PCR were employed to investigate the presence of other DNA viruses. Anal HPV infection was detected in 76.9% of the 230 MSM enrolled. The anal cytological reports were: 129 NILM, 37 ASC-US and 28 L-SIL (36 samples were inadequate for interpretation. HPV physical status was evaluated in the 109 anal specimens that harbored one or two different HPV genotypes. Integration was observed only in one HPV16-positive sample (0.9%, in which integrate-derived viral transcripts of type B were detected. Integration occurred in chromosome 14 q. In 22 of the 53 (41.5% mucosal HPV-negative samples, RCA restriction results would seem to indicate the presence of circular DNA viruses. Indeed, cutaneous HPV (4 samples, MCPyV (5 samples and TTV (4 samples were detected. In conclusion, anal HPV integration was rarely evidenced in HIV-uninfected MSM with no or mild anal cytological abnormalities, although the integration rate may have been underestimated because of the limitations of the employed assays. Other DNA viruses were detected in the anal samples of these individuals, although the significance of this occurrence needs to be assessed.

  10. Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors

    International Nuclear Information System (INIS)

    Pasquinelli, C.; Garreau, F.; Bougueleret, L.; Cariani, E.; Thiers, V.; Croissant, O.; Hadchouel, M.; Tiollais, P.; Brechot, C.; Grzeschik, K.H.

    1988-01-01

    Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. The authors have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possible related to HBV integration

  11. Presence of Mycobacterium leprae DNA and PGL-1 antigen in household contacts of leprosy patients from a hyperendemic area in Brazil.

    Science.gov (United States)

    Pinho, J D; Rivas, P M S; Mendes, M B P; Soares, R E P; Costa, G C; Nascimento, F R F; Paiva, M F L; Aquino, D M C; Figueireido, I A; Santos, A M; Pereira, S R F

    2015-11-19

    Leprosy is a highly infectious disease endemic to underdeveloped countries. In Maranhão State, Northeastern Brazil, the hyperendemic rate of 56.11 cases/100,000 inhabitants increased the necessity of better understanding the epidemiological profile of this population, particularly regarding efficient methods for evaluating individuals residing with diagnosed patients to understand disease transmission and the risk of infection. In this study, we examined the percentage of contacts with positive indices for Mycobacterium leprae DNA and phenol-glycolipid-1 antigen (PGL-1). PGL-1 was analyzed by an enzyme-linked immunosorbent assay, the ML-Flow test, and polymerase chain reaction of oral and nasal secretions of 808 leprosy contacts from Maranhão. PGL-1 was detected in 14.0% of patients and differed by operational classification of the index case (P leprae DNA in 5.6% of oral samples and 4.6% of nasal tissues, and 87% of subjects resided with high bacillary load patients. This study reinforces the efficacy of combining molecular and serological techniques to identify potential bacillus carriers in the asymptomatic stage of infection, such as in household contacts, highlighting the importance of these meth-ods for monitoring hyperendemic populations.

  12. Breast cancer proliferative activity: Is it the source of serum free DNA?

    African Journals Online (AJOL)

    Taha I. Hewala

    2013-04-13

    Apr 13, 2013 ... DNA concentration in lung cancer patients was 8-fold higher ... patients with invasive ductal carcinoma of clinical stages II and III,12 recently ... centrifuged for 20 min at 3500 rpm at 4 °C. The separated ser- um samples were ...

  13. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla F.; Olsen, Maja E.; Brandt, Luise Ørsted

    2011-01-01

    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  14. Estimation and quantification of human DNA in dental calculus: A pilot study.

    Science.gov (United States)

    Singh, Udita; Goel, Saurabh

    2017-01-01

    Identification using DNA has proved its accuracy multiple times in the field of forensic investigations. Investigators usually rely on either teeth or bone as the DNA reservoirs. However, there are instances where the skeletal or dental remains are not available or not preserved properly. Moreover, due to religious beliefs, the family members of the dead do not allow the investigating team to damage the remains for the sole purpose of identification. To investigate the presence of human DNA in dental calculus and to quantify the amount, if present. This prospective single-blinded pilot study included twenty subjects selected from the patients visiting a dental college. The samples of dental calculus were collected from the thickest portion of calculus deposited on the lingual surfaces of mandibular incisors. These samples were decontaminated and subjected to gel electrophoresis for DNA extraction. DNA was found in 85% cases. The amount of DNA varied from 21 to 37 μg/ml of dental calculus. Dental calculus is a rich reservoir of human DNA.

  15. Role of baseline HIV-1 DNA level in highly-experienced patients receiving raltegravir, etravirine and darunavir/ritonavir regimen (ANRS139 TRIO trial.

    Directory of Open Access Journals (Sweden)

    Charlotte Charpentier

    Full Text Available OBJECTIVE: In the ANRS 139 TRIO trial, the use of 3 new active drugs (raltegravir, etravirine, and darunavir/ritonavir, resulted in a potent and sustained inhibition of viral replication in multidrug-resistant treatment-experienced patients. The aim of this virological sub-study of the ANRS 139 TRIO trial was to assess: (i the evolution of HIV-1 DNA over the first year; and (ii the association between baseline HIV-1 DNA and virological outcome. METHODS: Among the 103 HIV-1-infected patients included in the ANRS-139 TRIO trial, HIV-1 DNA specimens were available for 92, 84, 88, and 83 patients at Week (W0, W12, W24, and W48, respectively. Quantification of total HIV-1 DNA was performed by using the commercial kit "Generic HIV DNA Cell" (Biocentric, Bandol, France. RESULTS: Baseline median HIV-1 DNA of patients displaying virological success (n= 61, viral blip (n= 20, and virological failure (n = 11 were 2.34 log(10 copies/10(6 PBMC (IQR= 2.15-2.66, 2.42 (IQR = 2.12-2.48, and 2.68 (IQR= 2.46-2.83, respectively. Although not statistically significant, patients exhibiting virological success or viral blip had a tendency to display lower baseline HIV-1 DNA than patients experiencing virological failure (P = 0.06. Median decrease of HIV-1 DNA between baseline and W48 was -0.13 log(10 copies/10(6 PBMC (IQR = -0.34 to +0.10, mainly explained by the evolution from W0 to W4. No more changes were observed in the W4-W48 period. CONCLUSIONS: In highly-experienced multidrug-resistant patients, HIV-1 DNA slightly decreased during the first month and then remained stable during the first year of highly potent antiretroviral regimen. In this population, baseline HIV-1 DNA might help to better predict the virological response and to tailor clinical therapeutic management as more aggressive therapeutic choices in patients with higher baseline HIV-1 DNA.

  16. At the forefront: evidence of the applicability of using environmental DNA to quantify the abundance of fish populations in natural lentic waters with additional sampling considerations

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    Klobucar, Stephen L.; Rodgers, Torrey W.; Budy, Phaedra

    2017-01-01

    Environmental DNA (eDNA) sampling has proven to be a valuable tool for detecting species in aquatic ecosystems. Within this rapidly evolving field, a promising application is the ability to obtain quantitative estimates of relative species abundance based on eDNA concentration rather than traditionally labor-intensive methods. We investigated the relationship between eDNA concentration and Arctic char (Salvelinus alpinus) abundance in five well-studied natural lakes; additionally, we examined the effects of different temporal (e.g., season) and spatial (e.g., depth) scales on eDNA concentration. Concentrations of eDNA were linearly correlated with char population estimates ( = 0.78) and exponentially correlated with char densities ( = 0.96 by area; 0.82 by volume). Across lakes, eDNA concentrations were greater and more homogeneous in the water column during mixis; however, when stratified, eDNA concentrations were greater in the hypolimnion. Overall, our findings demonstrate that eDNA techniques can produce effective estimates of relative fish abundance in natural lakes. These findings can guide future studies to improve and expand eDNA methods while informing research and management using rapid and minimally invasive sampling.

  17. Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases.

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    Marques, Lívia Érika Carlos; Frota, Cristiane Cunha; Quetz, Josiane da Silva; Bindá, Alexandre Havt; Mota, Rosa Maria Salane; Pontes, Maria Araci de Andrade; Gonçalves, Heitor de Sá; Kendall, Carl; Kerr, Ligia Regina Franco Sansigolo

    2017-12-26

    Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (T m  = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10 3 -8.02 × 10 5 , and in SB samples from MB patients were 1.87 × 10 3 -1.50 × 10 6 . Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

  18. Time Course of Detection of Human Male DNA from Stained Blood Sample on Various Surfaces by Loop Mediated Isothermal Amplification and Polymerase Chain Reaction

    Directory of Open Access Journals (Sweden)

    Panan Kanchanaphum

    2018-01-01

    Full Text Available This study explores determining the sex of humans from blood stains taken from different surfaces and compares the time course of detection with the conventional PCR, Conventional Loop Mediated Isothermal Amplification (LAMP, and LAMP-Lateral Flow Dipstick (LFD. For the DNA templates, 7 male and 7 female blood stained samples were extracted and added to LAMP and PCR reaction solution to amplify the SRY gene. The DNA samples were extracted from the following blood stained materials: cloth, wood, clay, and tile. Then, the samples were stored at room temperature for 1, 7, 30, and 60 day(s. After the DNA amplification, the gel electrophoresis process was applied to detect LAMP product. The LFD was combined with the LAMP to detect LAMP product on the male cloth samples. For the male samples, the time course of detection on the first and seventh days indicated positive for both LAMP and PCR products on all the surfaces while no DNA amplification was found on any of the female samples. On day 30, positive LAMP product was still found on all the male samples. However, it had faded on the tiles. Moreover, all the male samples, which had tested positive for PCR product, were blurred and unclear. On day 60, LAMP product was still found on all the male samples. Conversely, the PCR method resulted in no bands showing for any of the male samples. However, the LAMP-LFD method detected product on all the male samples of cloth. The results show that the LAMP is an effective, practical, and reliable molecular-biological method. Moreover, the LFD can increase the efficiency and sensitivity of the LAMP, making it more suitable for field studies because gel electrophoresis apparatus is not required.

  19. EGFR T790M mutation testing of non-small cell lung cancer tissue and blood samples artificially spiked with circulating cell-free tumor DNA: results of a round robin trial.

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    Fassunke, Jana; Ihle, Michaela Angelika; Lenze, Dido; Lehmann, Annika; Hummel, Michael; Vollbrecht, Claudia; Penzel, Roland; Volckmar, Anna-Lena; Stenzinger, Albrecht; Endris, Volker; Jung, Andreas; Lehmann, Ulrich; Zeugner, Silke; Baretton, Gustavo; Kreipe, Hans; Schirmacher, Peter; Kirchner, Thomas; Dietel, Manfred; Büttner, Reinhard; Merkelbach-Bruse, Sabine

    2017-10-01

    The European Commision (EC) recently approved osimertinib for the treatment of adult patients with locally advanced or metastatic non-small-cell lung cancer (NSCLC) harboring EGFR T790M mutations. Besides tissue-based testing, blood samples containing cell-free circulating tumor DNA (ctDNA) can be used to interrogate T790M status. Herein, we describe the conditions and results of a round robin trial (RRT) for T790M mutation testing in NSCLC tissue specimens and peripheral blood samples spiked with cell line DNA mimicking tumor-derived ctDNA. The underlying objectives of this two-staged external quality assessment (EQA) approach were (a) to evaluate the accuracy of T790M mutations testing across multiple centers and (b) to investigate if a liquid biopsy-based testing for T790M mutations in spiked blood samples is feasible in routine diagnostic. Based on a successfully completed internal phase I RRT, an open RRT for EGFR T790M mutation testing in tumor tissue and blood samples was initiated. In total, 48 pathology centers participated in the EQA. Of these, 47 (97.9%) centers submitted their analyses within the pre-defined time frame and 44 (tissue), respectively, 40 (plasma) successfully passed the test. The overall success rates in the RRT phase II were 91.7% (tissue) and 83.3% (blood), respectively. Thirty-eight out of 48 participants (79.2%) successfully passed both parts of the RRT. The RRT for blood-based EGFR testing initiated in Germany is, to the best of our knowledge, the first of his kind in Europe. In summary, our results demonstrate that blood-based genotyping for EGFR resistance mutations can be successfully integrated in routine molecular diagnostics complementing the array of molecular methods already available at pathology centers in Germany.

  20. Double positivity for HPV-DNA/p16ink4a is the biomarker with strongest diagnostic accuracy and prognostic value for human papillomavirus related oropharyngeal cancer patients.

    Science.gov (United States)

    Mena, Marisa; Taberna, Miren; Tous, Sara; Marquez, Sandra; Clavero, Omar; Quiros, Beatriz; Lloveras, Belen; Alejo, Maria; Leon, Xavier; Quer, Miquel; Bagué, Silvia; Mesia, Ricard; Nogués, Julio; Gomà, Montserrat; Aguila, Anton; Bonfill, Teresa; Blazquez, Carmen; Guix, Marta; Hijano, Rafael; Torres, Montserrat; Holzinger, Dana; Pawlita, Michael; Pavon, Miguel Angel; Bravo, Ignacio G; de Sanjosé, Silvia; Bosch, Francesc Xavier; Alemany, Laia

    2018-03-01

    The etiologic role of human papillomaviruses (HPV) in oropharyngeal cancer (OPC) is well established. Nevertheless, information on survival differences by anatomic sub-site or treatment remains scarce, and it is still unclear the HPV-relatedness definition with best diagnostic accuracy and prognostic value. We conducted a retrospective cohort study of all patients diagnosed with a primary OPC in four Catalonian hospitals from 1990 to 2013. Formalin-fixed, paraffin-embedded cancer tissues were subjected to histopathological evaluation, DNA quality control, HPV-DNA detection, and p16 INK4a /pRb/p53/Cyclin-D1 immunohistochemistry. HPV-DNA positive and a random sample of HPV-DNA negative cases were subjected to HPV-E6*I mRNA detection. Demographic, tobacco/alcohol use, clinical and follow-up data were collected. Multivariate models were used to evaluate factors associated with HPV positivity as defined by four different HPV-relatedness definitions. Proportional-hazards models were used to compare the risk of death and recurrence among HPV-related and non-related OPC. 788 patients yielded a valid HPV-DNA result. The percentage of positive cases was 10.9%, 10.2%, 8.5% and 7.4% for p16 INK4a , HPV-DNA, HPV-DNA/HPV-E6*I mRNA, and HPV-DNA/p16 INK4a , respectively. Being non-smoker or non-drinker was consistently associated across HPV-relatedness definitions with HPV positivity. A suggestion of survival differences between anatomic sub-sites and treatments was observed. Double positivity for HPV-DNA/p16 INK4a showed strongest diagnostic accuracy and prognostic value. Double positivity for HPV-DNA/p16 INK4a , a test that can be easily implemented in the clinical practice, has optimal diagnostic accuracy and prognostic value. Our results have strong clinical implications for patients' classification and handling and also suggest that not all the HPV-related OPC behave similarly. Copyright © 2018 Elsevier Ltd. All rights reserved.