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Sample records for hpv dna extraction

  1. Efficient DNA extraction for HPV genotyping in formalin-fixed, paraffin-embedded tissues.

    Science.gov (United States)

    Steinau, Martin; Patel, Sonya S; Unger, Elizabeth R

    2011-07-01

    DNA from archived FFPE can be used for papillomavirus genotyping, but potential problems include paraffin as a physical barrier, DNA cross-linking, and PCR inhibitors. To address these complications, we combined a commercially available DNA isolation kit (Qiagen DNeasy) with a heat treatment and evaluated the resulting DNA with regards to HPV typing. DNA was extracted from 10-μm sections from 150 FFPE cancer samples. One protocol followed the manufacturer's recommendation, including paraffin removal by xylene and tissue lysis at 56°C. A second section was directly incubated at 120°C and subsequently lysed at 65°C. After spin-column purification, both extracts were tested with a linear array HPV genotyping assay. Additionally, cellular DNA yield, HPV16 DNA copies, and PCR inhibitors were assessed by real-time qPCR assays. Inadequate linear array HPV genotyping assay results were significantly more frequent (P = 0.0003) in xylene-treated (29/150, 19.3%) than in heat-treated extracts (8/150, 5.3%). HPV detection also differed, with 94/150 (62.7%) and 110/150 (73.3%) positive results, respectively (P = 0.0026). The heat method also yielded more PCR-amplifiable cellular DNA (8.2-fold; P protocols, resulting in higher DNA yields and increased sensitivity for HPV testing.

  2. HPV DNA test

    Science.gov (United States)

    ... is generally not recommended for detecting low-risk HPV infections. ... Human papilloma virus - testing; Abnormal Pap smear - HPV testing; LSIL-HPV testing, Low-grade dysplasia - HPV testing; HSIL - HPV testing; High-grade dysplasia - HPV testing; HPV ...

  3. Comparative evaluation of different DNA extraction methods for HPV genotyping by linear array and INNO-LiPA.

    Science.gov (United States)

    Donà, Maria Gabriella; Benevolo, Maria; Pimpinelli, Fulvia; Battista, Mara; Rollo, Francesca; Stivali, Francesca; Moscarelli, Antonella; Giuliani, Massimo; Di Carlo, Aldo; Vocaturo, Amina

    2011-06-01

    In order to investigate the influence of DNA extraction on two PCR-based HPV genotyping tests (Linear Array, Roche and INNO-LiPA Extra, Innogenetics), three different procedures were used to purify DNA from 28 cervico-vaginal samples tested previously by the Hybrid Capture 2: the AmpliLute Liquid Media Extraction kit (Roche), the QIAamp DNA Blood mini kit (QIAGEN), and the NucliSENS EasyMAG automated platform (bioMérieux). All HC2-positive samples were found positive by both assays, independently of the extract used. Type-specific concordance (i.e., identical HPV type-specific profile in all the extracts of the same sample) was observed in 55% and 75% of the cases testing samples by the Linear Array and the INNO-LiPA, respectively. Using the DNA extracted with the two manual methods the results were concordant in 75% of the cases both for the Linear Array and the INNO-LiPA. When comparing the Linear Array results obtained on either of the two manual extracts with those obtained following automated extraction, 65% of the samples showed type-specific concordance in both cases. The INNO-LiPA results were concordant in 80% of the cases comparing the AmpliLute versus the automated extract, while concordant results were observed in 90% of the cases when comparing the QIAGEN versus the automated extract. In conclusion, the Linear Array and INNO-LiPA results are affected by the method of DNA extraction. Consequently, different HPV type-specific profiles may be observed using different extracts of the same sample. The use of consistent protocols for DNA purification is a priority to guarantee intra-assay reproducibility over time.

  4. Comparison of different methods for extraction and purification of human Papillomavirus (HPV) DNA from serum samples

    Science.gov (United States)

    Azizah, N.; Hashim, U.; Nadzirah, Sh.; Arshad, M. K. Md; Ruslinda, A. R.; Gopinath, Subash C. B.

    2017-03-01

    The affectability and unwavering quality of PCR for indicative and research purposes require effective fair systems of extraction and sanitization of nucleic acids. One of the real impediments of PCR-based tests is the hindrance of the enhancement procedure by substances exhibit in clinical examples. This examination considers distinctive techniques for extraction and cleaning of viral DNA from serum tests in view of recuperation productivity as far as yield of DNA and rate recouped immaculateness of removed DNA, and rate of restraint. The best extraction strategies were the phenol/chloroform strategy and the silica gel extraction methodology for serum tests, individually. Considering DNA immaculateness, extraction technique by utilizing the phenol/chloroform strategy delivered the most tasteful results in serum tests contrasted with the silica gel, separately. The nearness of inhibitors was overcome by all DNA extraction strategies in serum tests, as confirm by semiquantitative PCR enhancement.

  5. Effect of different concentration of HPV DNA probe immobilization for cervical cancer detection based IDE biosensor

    Science.gov (United States)

    Roshila, M. L.; Hashim, U.; Azizah, N.; Nadzirah, Sh.; Arshad, M. K. Md; Ruslinda, A. R.; Gopinath, Subash C. B.

    2017-03-01

    This paper principally delineates to the detection process of Human Papillomavirus (HPV) DNA test. HPV is an extremely common virus infection that infected to human by the progressions cell in the cervix cell. The types of HPV that give a most exceedingly awful infected with cervical cancer is 16 and 18 other than 31 and 45. The HPV DNA probe is immobilized with a different concentration to stabilize the sensitivity. A technique of rapid and sensitive for the HPV identification was proposed by coordinating basic DNA extraction with a quality of DNA. The extraction of the quality of DNA will make a proficiency of the discovery procedure. It will rely on the sequence of the capture probes and the way to support their attached. The fabrication, surface modification, immobilization and hybridization procedures are described by current-voltage (I-V) estimation by utilizing KEITHLEY 6487. This procedure will play out a decent affectability and selectivity of HPV discovery.

  6. HPV16 detection by qPCR method in relation to quantity and quality of DNA extracted from archival formalin fixed and paraffin embedded head and neck cancer tissues by three commercially available kits.

    Science.gov (United States)

    Biesaga, Beata; Janecka, Anna; Mucha-Małecka, Anna; Adamczyk, Agnieszka; Szostek, Sława; Słonina, Dorota; Halaszka, Krzysztof; Przewoźnik, Marcin

    2016-10-01

    The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically. Its quality was analyzed using A260/280 and A260/230 ratios and the β-actin fragment amplifiability in qPCR. HPV16 presence was detected by qPCR, using specific primers and TaqMan probe. HPV infection was found in 8 DNA samples extracted with M kit (38.1%) and in 7 (33.3%) isolated with Q and P kits. Three samples from M and Q kits were characterized by HPV16 positivity and lack of β-actin amplifiability. They had significantly lower A260/280 ratio (M: 1.6±0.0, p=0.044 and Q: 1.7±0.0, p=0.016) compared to samples with both fragments amplification (M: 1.7±0.0 and Q: 1.9±0.0). Therefore, for HPV detection by qPCR in FFPE tissues we recommend ReliaPrep™ FFPE gDNA Miniprep System.

  7. Study of concentration of HPV DNA probe immobilization for cervical cancer detection based IDE biosensor

    Science.gov (United States)

    Roshila, M. L.; Hashim, U.; Azizah, N.

    2016-07-01

    This paper mainly illustrates regarding the detection process of Human Papillomavirus (HPV) DNA probe. HPV is the most common virus that infected to human by a sexually transmitted virus. The most common high-risk HPV are 16 and 18. Interdigitated electrode (IDE) device used as based of Titanium Dioxide (TiO2) acts as inorganic surface, where by using APTES as a linker between inorganic surface and organic surface. A strategy of rapid and sensitive for the HPV detection was proposed by integrating simple DNA extraction with a gene of DNA. The extraction of the gene of DNA will make an efficiency of the detection process. It will depend on the sequence of the capture probes and the way to support their attached. The fabrication, surface modification, immobilization and hybridization processes are characterized by current voltage (I-V) measurement by using KEITHLEY 6487. This strategy will perform a good sensitivity of HPV detection.

  8. HPV 9G DNA Chip: 100% Clinical Sensitivity and Specificity

    OpenAIRE

    An, Heejung; Song, Keum-Soo; Nimse, Satish Balasaheb; Kim, Junghoon; Nguyen, Van-Thuan; Ta, Van-Thao; Sayyed, Danishmalik Rafiq; Kim, Taisun

    2012-01-01

    We describe a novel HPV 9G DNA chip test for the accurate and reliable genotyping of human papillomavirus (HPV). The HPV 9G DNA chip test established its efficiency in terms of a signal-to-background ratio (SBR) of 200, which is 50 times superior to commercial HPV DNA chips, and 100% target-specific hybridization at 25°C. We compared the genotyping results for the 439 clinical samples by the HPV 9G DNA chip test with the sequencing results for the MY11/GP6+ (M2) primer set-mediated PCR produc...

  9. Comparison of HPV detection technologies: Hybrid capture 2, PreTect HPV-Proofer and analysis of HPV DNA viral load in HPV16, HPV18 and HPV33 E6/E7 mRNA positive specimens.

    LENUS (Irish Health Repository)

    Keegan, Helen

    2012-02-01

    Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6\\/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205\\/299) of the cases were positive by hc2 and 38% (112\\/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease.

  10. HPV Genotyping from the high risk mRNA Aptima assay- a direct approach using DNA from Aptima sample tubes.

    Science.gov (United States)

    Lillsunde Larsson, G; Kaliff, M; Bergengren, L; Karlsson, M G; Helenius, G

    2016-09-01

    The underlying cause of cervical cancer is an infection with the human papilloma virus (HPV) and HPV testing can be used for cervical cancer screening. The Aptima HPV assay from Hologic is an mRNA HPV test used to identify clinically relevant infections but the method does not discriminate between the different high risk genotypes. The aim of the current study was to evaluate if analyzed Aptima sample transfer tubes could be used as a source for HPV genotyping, using sample DNA. Study samples (n=108); were HPV-tested with mRNA Aptima assay and in parallel DNA was extracted and genotyped with Anyplex II HPV28. Analyzed mRNA Aptima tubes were thereafter used as source for a second DNA extraction and genotyping. Using mRNA Aptima result as reference, 90% of the samples (35/39) were high risk positive with the Anyplex II HPV28. Cohen's kappa 0.78 (95% CI: 0.66-0.90), sensitivity 0.90 (95% CI: 0.76-0.97) and specificity 0.90 (95% CI: 0.80-0.96). Two discordant samples carried low-risk genotypes (HPV 82 and HPV 44) and two were negative. DNA-genotyping results, in parallel to and after mRNA testing, were compared and differed significantly (McNemar test: P=0.021) possibly due to sample extraction volume difference. Cohen's kappa 0.81 (95% CI: 0.70-0.92), sensitivity 0.85 (95% CI: 0.74-0.93) and specificity 0.98 (95% CI: 0.88-1.00). In conclusion, analyzed mRNA Aptima sample tubes could be used as a source for DNA HPV genotyping. The sample volume used for extraction needs to be further explored.

  11. Comparing triage algorithms using HPV DNA genotyping, HPV E7 mRNA detection and cytology in high-risk HPV DNA-positive women.

    Science.gov (United States)

    Luttmer, Roosmarijn; Berkhof, Johannes; Dijkstra, Maaike G; van Kemenade, Folkert J; Snijders, Peter J F; Heideman, Daniëlle A M; Meijer, Chris J L M

    2015-06-01

    High-risk human papillomavirus (hrHPV) DNA positive women require triage testing to identify those with high-grade cervical intraepithelial neoplasia or cancer (≥CIN2). Comparing three triage algorithms (1) E7 mRNA testing following HPV16/18/31/33/45/52/58 genotyping (E7 mRNA test), (2) HPV16/18 DNA genotyping and (3) cytology, for ≥CIN2 detection in hrHPV DNA-positive women. hrHPV DNA-positive women aged 18-63 years visiting gynecology outpatient clinics were included in a prospective observational cohort study. From these women a cervical scrape and colposcopy-directed biopsies were obtained. Cervical scrapes were evaluated by cytology, HPV DNA genotyping by bead-based multiplex genotyping of GP5+6+-PCR-products, and presence of HPV16/18/31/33/45/52/58 E7 mRNA using nucleic acid sequence-based amplification (NASBA) in DNA positive women for respective HPV types. Sensitivities and specificities for ≥CIN2 were compared between E7 mRNA test and HPV16/18 DNA genotyping in the total group (n=348), and E7 mRNA test and cytology in a subgroup of women referred for non-cervix-related gynecological complaints (n=133). Sensitivity for ≥CIN2 of the E7 mRNA test was slightly higher than that of HPV16/18 DNA genotyping (66.9% versus 60.9%; ratio 1.10, 95% CI: 1.0002-1.21), at similar specificity (54.8% versus 52.3%; ratio 1.05, 95% CI: 0.93-1.18). Neither sensitivity nor specificity of the E7 mRNA test differed significantly from that of cytology (sensitivity: 68.8% versus 75.0%; ratio 0.92, 95% CI: 0.72-1.17; specificity: 59.4% versus 65.3%; ratio 0.91, 95% CI: 0.75-1.10). For detection of ≥CIN2 in hrHPV DNA-positive women, an algorithm including E7 mRNA testing following HPV16/18/31/33/45/52/58 DNA genotyping performs similar to HPV16/18 DNA genotyping or cytology. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination.

    Science.gov (United States)

    Marcuzzi, Gian Paolo; Awerkiew, Sabine; Hufbauer, Martin; Schädlich, Lysann; Gissmann, Lutz; Eming, Sabine; Pfister, Herbert

    2014-06-01

    The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

  13. Clinical Significance of an HPV DNA Chip Test with Emphasis on HPV-16 and/or HPV-18 Detection in Korean Gynecological Patients

    Science.gov (United States)

    Yeo, Min-Kyung; Lee, Ahwon; Hur, Soo Young; Park, Jong Sup

    2016-01-01

    Background: Human papillomavirus (HPV) is a major risk factor for cervical cancer. Methods: We evaluated the clinical significance of the HPV DNA chip genotyping assay (MyHPV chip, Mygene Co.) compared with the Hybrid Capture 2 (HC2) chemiluminescent nucleic acid hybridization kit (Digene Corp.) in 867 patients. Results: The concordance rate between the MyHPV chip and HC2 was 79.4% (kappa coefficient, κ = 0.55). The sensitivity and specificity of both HPV tests were very similar (approximately 85% and 50%, respectively). The addition of HPV result (either MyHPV chip or HC2) to cytology improved the sensitivity (95%, each) but reduced the specificity (approximately 30%, each) compared with the HPV test or cytology alone. Based on the MyHPV chip results, the odds ratio (OR) for ≥ high-grade squamous intraepithelial lesions (HSILs) was 9.9 in the HPV-16/18 (+) group and 3.7 in the non-16/18 high-risk (HR)-HPV (+) group. Based on the HC2 results, the OR for ≥ HSILs was 5.9 in the HR-HPV (+) group. When considering only patients with cytological diagnoses of “negative for intraepithelial lesion or malignancy” and “atypical squamous cell or atypical glandular cell,” based on the MyHPV chip results, the ORs for ≥ HSILs were 6.8 and 11.7, respectively, in the HPV-16/18 (+) group. Conclusions: The sensitivity and specificity of the MyHPV chip test are similar to the HC2. Detecting HPV-16/18 with an HPV DNA chip test, which is commonly used in many Asian countries, is useful in assessing the risk of high-grade cervical lesions. PMID:27345180

  14. Clinical Significance of an HPV DNA Chip Test with Emphasis on HPV-16 and/or HPV-18 Detection in Korean Gynecological Patients

    Directory of Open Access Journals (Sweden)

    Min-Kyung Yeo

    2016-07-01

    Full Text Available Background: Human papillomavirus (HPV is a major risk factor for cervical cancer. Methods: We evaluated the clinical significance of the HPV DNA chip genotyping assay (MyHPV chip, Mygene Co. compared with the Hybrid Capture 2 (HC2 chemiluminescent nucleic acid hybridization kit (Digene Corp. in 867 patients. Results: The concordance rate between the MyHPV chip and HC2 was 79.4% (kappa coefficient, κ = 0.55. The sensitivity and specificity of both HPV tests were very similar (approximately 85% and 50%, respectively. The addition of HPV result (either MyHPV chip or HC2 to cytology improved the sensitivity (95%, each but reduced the specificity (approximately 30%, each compared with the HPV test or cytology alone. Based on the MyHPV chip results, the odds ratio (OR for ≥ high-grade squamous intraepithelial lesions (HSILs was 9.9 in the HPV-16/18 (+ group and 3.7 in the non-16/18 high-risk (HR-HPV (+ group. Based on the HC2 results, the OR for ≥ HSILs was 5.9 in the HR-HPV (+ group. When considering only patients with cytological diagnoses of “negative for intraepithelial lesion or malignancy” and “atypical squamous cell or atypical glandular cell,” based on the MyHPV chip results, the ORs for ≥ HSILs were 6.8 and 11.7, respectively, in the HPV-16/18 (+ group. Conclusions: The sensitivity and specificity of the MyHPV chip test are similar to the HC2. Detecting HPV-16/18 with an HPV DNA chip test, which is commonly used in many Asian countries, is useful in assessing the risk of high-grade cervical lesions.

  15. HPV Direct Flow CHIP: a new human papillomavirus genotyping method based on direct PCR from crude-cell extracts.

    Science.gov (United States)

    Herraez-Hernandez, Elsa; Alvarez-Perez, Martina; Navarro-Bustos, Gloria; Esquivias, Javier; Alonso, Sonia; Aneiros-Fernandez, Jose; Lacruz-Pelea, Cesar; Sanchez-Aguera, Magdalena; Santamaria, Javier Saenz; de Antonio, Jesus Chacon; Rodriguez-Peralto, Jose Luis

    2013-10-01

    HPV Direct Flow CHIP is a newly developed test for identifying 18 high-risk and 18 low-risk human papillomavirus (HPV) genotypes. It is based on direct PCR from crude-cell extracts, automatic flow-through hybridization, and colorimetric detection. The aim of this study was to evaluate the performance of HPV Direct Flow CHIP in the analysis of 947 samples from routine cervical screening or the follow-up of abnormal Pap smears. The specimens were dry swab samples, liquid-based cytology samples, or formalin-fixed paraffin-embedded tissues. The genotype distribution was in agreement with known epidemiological data for the Spanish population. Three different subgroups of the samples were also tested by Linear Array (LA) HPV Genotyping Test (n=108), CLART HPV2 (n=82), or Digene Hybrid Capture 2 (HC2) HPV DNA Test (n=101). HPV positivity was 73.6% by HPV Direct Flow CHIP versus 67% by LA, 65.9% by HPV Direct Flow CHIP versus 59.8% by CLART, and 62.4% by HPV Direct Flow CHIP versus 42.6% by HC2. HPV Direct Flow CHIP showed a positive agreement of 88.6% with LA (k=0.798), 87.3% with CLART (k=0.818), and 68.2% with HC2 (k=0.618). In conclusion, HPV Direct Flow CHIP results were comparable with those of the other methods tested. Although further investigation is needed to compare the performance of this new test with a gold-standard reference method, these preliminary findings evidence the potential value of HPV Direct Flow CHIP in HPV vaccinology and epidemiology studies.

  16. Polymerase chain reaction and conventional DNA tests in detection of HPV DNA in cytologically normal and abnormal cervical scrapes

    DEFF Research Database (Denmark)

    Kalia, A.; Jalava, T.; Nieminen, P.

    1992-01-01

    Med.mikrobiologi, polymerase chain reaction, DNA tests, human papillomavirus (HPV), cervical smear, hybridisation, cytologi, affiProbe HPV test, ViraType test......Med.mikrobiologi, polymerase chain reaction, DNA tests, human papillomavirus (HPV), cervical smear, hybridisation, cytologi, affiProbe HPV test, ViraType test...

  17. High quality DNA from human papillomavirus (HPV for PCR/RFLPs

    Directory of Open Access Journals (Sweden)

    Denise Wanderlei-Silva

    2005-01-01

    Full Text Available The analysis of DNA in clinical samples for a secure diagnostic has become indispensable nowadays. Techniques approaching isolation of high molecular weigth DNA of HPV could lead to efficient amplification and early clinical diagnosis of the virus DNA by PCR (polymerase chain reaction. We describe a fast, non-toxical, efficient and cheap method for DNA isolation of human papilloma virus (HPV from cervical smears using guanidine (DNAzol solution. A 450 bp DNA band correponding to the late region (L1 of the virus genome was detected by PCR, showing that the DNAzol extraction soluction generated a good viral DNA yield. The electrophoretic pattern after digestion with restriction endonucleases (RFLPs/PCR revealed the predominance of HPV-16 and HPV-33 in the samples from the State of Alagoas, Brazil.A detecção de DNA em amostras clínicas visando um diagnóstico mais seguro vem se tornando uma prática comum em laboratórios de análise clínica. Metodologias que objetivem o isolamento de DNA de alto peso molecular de HPV podem levar a uma amplificação precisa e diagnose precoce do DNA do vírus por PCR (reação de polimerase em cadeia. Nós descrevemos um método para o isolamento do DNA do vírus do papiloma humano de amostras cervicais utilizando o detergente guanidina (solução DNAzol. O método foi rápido, não-tóxico e eficiente. Uma banda de DNA de 450 pb correspondente à região tardia (L1 do genoma viral foi detectada por PCR, mostrando que a extração com DNAzol gerou quantidade suficiente de DNA para análise. O padrão eletroforético, após digestão com endonucleases de restrição (RFLPs/PCR, revelou predominância de HPV 16 e HPV-33 nas amostras no Estado de Alagoas, Brasil.

  18. Detection of HPV DNA and immunohistochemical expression of cell cycle proteins in oral carcinoma in a population of brazilian patients

    Directory of Open Access Journals (Sweden)

    Rosilene Calazans Soares

    2008-10-01

    Full Text Available This study investigated the presence of human papillomavirus (HPV DNA and viral types in 33 cases of oral squamous cells carcinoma (OSCC and compared the immunohistochemical expression of the cell-cycle markers p21 and pRb between cases of the disease with and without HPV. DNA was extracted from paraffin-embedded tissue and amplified by PCR for the detection of HPV DNA. Viral typing was performed by dot blot hybridization. Immunohistochemistry was performed by the streptavidinbiotin technique. HPV DNA was detected in 11 (33.33% of the 33 cases. The prevalent viral type was HPV 18 (81.81%. A significant association was observed between the presence of HPV and immunohistochemical expression of pRb, but not between p21 expression and the presence of the virus. The low frequency of detection of HPV DNA in OSCC suggests a possible participation of the virus in the development and progression of only a subgroup of these tumors.

  19. HPV

    Science.gov (United States)

    Human papillomaviruses (HPV) are common viruses that can cause warts. There are more than 100 types of HPV. Most are harmless, but about 30 types put ... either low-risk or high-risk. Low-risk HPV can cause genital warts. High-risk HPV can ...

  20. Comparison of the Cobas 4800 HPV and HPV 9G DNA Chip Tests for Detection of High-Risk Human Papillomavirus in Cervical Specimens of Women with Consecutive Positive HPV Tests But Negative Pap Smears.

    Science.gov (United States)

    Jun, Sun-Young; Park, Eun Su; Kim, Jiyoung; Kang, Jun; Lee, Jae Jun; Bae, Yoonjin; Kim, Sang-Il; Maeng, Lee-So

    2015-01-01

    Detecting high-risk (HR) HPV is important for clinical management of women with persistent HPV-positive and Pap-negative results. The Cobas 4800 HPV test is the first FDA-approved HPV DNA test that can be used alone as a first-line screening tool. The HPV 9G DNA chip test is a PCR-based DNA microarray assay. We evaluated the patients of consecutive HPV-positivity on HPV 9G DNA chip test without cytologic abnormalities. We then compared the performances of HPV 9G DNA chip and the Cobas 4800 HPV tests for detecting HR HPV with each other and confirmed HPV genotyping using direct sequencing. All 214 liquid-based cytology specimens were collected from 100 women with consecutive HPV-positive and Pap-negative results on the HPV 9G DNA chip test between May 2012 and Dec 2013, but only 180 specimens were available for comparing HPV test results. The HPV 9G DNA chip and the Cobas 4800 HPV tests agreed with each other in 81.7% of the samples, and the concordance rate was greater than 97.2% for detecting HPV-16 or -18. For HR genotypes other than HPV types 16 and 18, the two tests agreed for 81.1% of the samples. The sensitivity of both assays for detecting HR HPV was 100%, regardless of HR genotypes. The HPV 9G DNA chip test may be as effective as the Cobas 4800 HPV test in detecting HR HPV, and has a similar ability to identify HPV-16 and -18.

  1. Unscheduled DNA replication origin activation at inserted HPV 18 sequences in a HPV-18/MYC amplicon.

    Science.gov (United States)

    Conti, Chiara; Herrick, John; Bensimon, Aaron

    2007-08-01

    Oncogene amplification is a critical step leading to tumorigenesis, but the underlying mechanisms are still poorly understood. Despite data suggesting that DNA replication is a major source of genomic instability, little is known about replication origin usage and replication fork progression in rearranged regions. Using a single DNA molecule approach, we provide here the first study of replication kinetics on a previously characterized MYC/papillomavirus (HPV18) amplicon in a cervical cancer. Using this amplicon as a model, we investigated the role DNA replication control plays in generating amplifications in human cancers. The data reveal severely perturbed DNA replication kinetics in the amplified region when compared with other regions of the same genome. It was found that DNA replication is initiated from both genomic and viral sequences, resulting in a higher median frequency of origin firings. In addition, it was found that the higher initiation frequency was associated with an equivalent increase in the number of stalled replication forks. These observations raise the intriguing possibility that unscheduled replication origin activation at inserted HPV-18 viral DNA sequences triggers DNA amplification in this cancer cell line and the subsequent overexpression of the MYC oncogene.

  2. Identification of human papillomavirus (HPV) 16 DNA integration and the ensuing patterns of methylation in HPV-associated head and neck squamous cell carcinoma cell lines.

    Science.gov (United States)

    Hatano, Takashi; Sano, Daisuke; Takahashi, Hideaki; Hyakusoku, Hiroshi; Isono, Yasuhiro; Shimada, Shoko; Sawakuma, Kae; Takada, Kentaro; Oikawa, Ritsuko; Watanabe, Yoshiyuki; Yamamoto, Hiroyuki; Itoh, Fumio; Myers, Jeffrey N; Oridate, Nobuhiko

    2017-04-01

    Recent studies showed that human papillomavirus (HPV) integration contributes to the genomic instability seen in HPV-associated head and neck squamous cell carcinoma (HPV-HNSCC). However, the epigenetic alterations induced after HPV integration remains unclear. To identify the molecular details of HPV16 DNA integration and the ensuing patterns of methylation in HNSCC, we performed next-generation sequencing using a target-enrichment method for the effective identification of HPV16 integration breakpoints as well as the characterization of genomic sequences adjacent to HPV16 integration breakpoints with three HPV16-related HNSCC cell lines. The DNA methylation levels of the integrated HPV16 genome and that of the adjacent human genome were also analyzed by bisulfite pyrosequencing. We found various integration loci, including novel integration sites. Integration loci were located predominantly in the intergenic region, with a significant enrichment of the microhomologous sequences between the human and HPV16 genomes at the integration breakpoints. Furthermore, various levels of methylation within both the human genome and the integrated HPV genome at the integration breakpoints in each integrant were observed. Allele-specific methylation analysis suggested that the HPV16 integrants remained hypomethylated when the flanking host genome was hypomethylated. After integration into highly methylated human genome regions, however, the HPV16 DNA became methylated. In conclusion, we found novel integration sites and methylation patterns in HPV-HNSCC using our unique method. These findings may provide insights into understanding of viral integration mechanism and virus-associated carcinogenesis of HPV-HNSCC.

  3. Clinical significance of HPV-DNA testing for precancerous cervical lesionS

    OpenAIRE

    Moarcăs, M; Georgescu, IC; Brătilă, E; Badea, M.; Cîrstoiu, ECM

    2014-01-01

    Cervical screening by using cytology was proven efficient in reducing the mortality secondary to cervical cancer, but this method has limitations. High risk HPV infection is essential for cervical cancer development so HPV testing is a new tool used for screening patients for cervical neoplasia. HPV testing was proven most useful for women over 30 years old, in cases in which cytology identified ASC-US and after treatment for CIN. This article outlines the clinical significance of HPV-DNA tes...

  4. Determinants of seropositivity among HPV-16/18 DNA positive young women

    Directory of Open Access Journals (Sweden)

    Porras Carolina

    2010-08-01

    Full Text Available Abstract Background Not all women infected with HPV-16/18 have detectable levels of HPV-16/18 antibodies, those who seroconvert develop low antibody levels, and seroconversion occurs typically several months post-infection. We evaluated determinants of seropositivity among 646 women infected with HPV-16 and/or HPV-18. Methods Data are from the enrollment visit of the NCI-sponsored Costa Rica HPV Vaccine Trial. Sera were tested for HPV-16/18 antibodies by ELISA; cervical specimens were tested for HPV DNA using HC2 and SPF10/LiPA25. Odds ratios (OR and 95% confidence intervals (CI were computed. Results Among HPV-16/18 DNA positives, seropositivity was 63.0% and 57.5%, respectively. Among HPV-16 DNA positives, seropositivity increased with lifetime number of sexual partners (p-trend = 0.01. Women with abnormal cytology and/or high viral load had a 1.63-2.79-fold increase in the detection of antibodies compared to women with normal cytology/low viral load. Current users of oral contraceptives had a 1.88-fold (95%CI, 1.14-3.09 increased detection of antibodies and current users of injectables had a 3.38-fold (95%CI, 1.39-8.23 increased detection compared to never users. Among HPV-18 DNA positive women, seropositivity was associated with current oral contraceptive use (OR 2.47; 95%CI 1.08-5.65. Conclusions Factors associated with sustained HPV exposure (abnormal cytology, elevated HPV viral load, increasing lifetime partners were predictive of HPV-16 seropositivity. Hormonal contraceptive use was associated with seropositivity suggesting an effect of hormones on immune responses to HPV. Patterns were less consistent for HPV-18. Follow up of incident HPV infections to evaluate seroconversion and their determinants is needed.

  5. Human papillomavirus (HPV types 16, 18, 31, 45 DNA loads and HPV-16 integration in persistent and transient infections in young women

    Directory of Open Access Journals (Sweden)

    Ferenczy Alex

    2010-11-01

    Full Text Available Abstract Background HPV burden is a predictor for high-grade cervical intraepithelial neoplasia and cancer. The natural history of HPV load in young women being recently exposed to HPV is described in this paper. Methods A total of 636 female university students were followed for 2 years. Cervical specimens with HPV-16, -18, -31, or -45 DNA by consensus PCR were further evaluated with type-specific and β-globin real-time PCR assays. Proportional hazards regression was used to estimate hazard ratios (HR of infection clearance. Generalized estimating equations assessed whether HPV loads was predictive of HPV infection at the subsequent visit. Results HPV loads were consistently higher among women Conclusions The association between HPV load and persistence is not uniform across high-risk genital genotypes. HPV-16 integration was only rarely demonstrated in young women.

  6. Expression of HPV-DNA in retinoblastoma tissues%视网膜母细胞瘤组织中 HPV-DNA 表达的研究

    Institute of Scientific and Technical Information of China (English)

    刘国颖; 杨华胜

    2013-01-01

    目的:研究我国视网膜母细胞瘤(retinoblastoma,RB)中HPV-DNA表达情况,为进一步揭示HPV 在散发性RB中作用提供初步依据。方法:收集22例散发性RB患者新鲜肿瘤组织标本,应用PCR方法及引物MY09/11检测肿瘤组织中HPV-DNA表达情况。结果:选取22例RB 肿瘤标本中7例HPV-DNA阳性(32%),均为单眼散发患者(包括男4例,女3例),2例双眼散发患者均为阴性。结论:HPV可能是我国散发性RB发病因素之一,但其明确作用及机制尚待进一步研究。%AIM: To investigate the expression of HPV-DNA in retinoblastoma tumor samples in China, and to provide preliminary evidence for the role of HPV in sporadic retinoblastoma ( RB) . METHODS: Fresh tumor tissues from 22 children with sporadic RB were collected to detect the presence of HPV-DNA using polymerase chain reaction ( PCR ) and MY09/11 primers. RESULTS: HPV-DNA was present in 7/22 ( 32%) RB tumors.All the positive cases were sporadic unilateral RB ( 4 cases were male, 3 cases were female ) . Two bilateral RB cases were negative for HPV-DNA. CONCLUSION:The presence of HPV in RB tumors can be a pathogenic factor for sporadic RB.But its role and mechanism in carcinogenesis needs further research.

  7. Value and Feasibility of HPV DNA Test in Cervical Scraping Smears

    Institute of Scientific and Technical Information of China (English)

    WU Sufang; LIAO Guoning; MA Ding; CHEN Gang; WANG Wei; XU Qian; GU Hainian; LU Yunping; ZHOU Liping; DU Juan; LI Fujun

    2005-01-01

    To investigate the reliability and feasibility of human papillomavirus (HPV) DNA test in cervical scraping smears with polymerase chain reaction (PCR), 131 cases of cervical scraping specimens were collected, and the positive rates and accuracy of HPV infection were determined in normal subjects and cervical cancer patients. GP5+/GP6+ and E7 primer pairs designed for detecting HPV L1 and HPV type 16 E7 were tested in this study. Our results showed that positive rates of HPV DNA in normal population and cervical cancer patients were 32.99 % and 73.53 % respectively and there was significant difference between them (P<0. 001). In normal subjects, detection rates of HPV DNA with GP5+/GP6+ and E7 primer pairs were 27.84 % and 16.49 % respectively, with statistically significant difference between them (P>0.05). However the detection rates in cervical cancer patients were 38.24 % and 67.65 % for the two markers, with a significant difference found between them (P<0.05). It is concluded that HPV DNA test with PCR for cervical scraping smears was feasible. GP5+/GP6+ primer pairs may be a useful probe to screen HPV infection in normal population, but they are not sensitive enough in cervical cancer patients. It is suggested that high risk type HPV DNA test was very useful in population with high risk of cervical cancer.

  8. Fusion of CTLA-4 with HPV16 E7 and E6 enhanced the potency of therapeutic HPV DNA vaccine.

    Directory of Open Access Journals (Sweden)

    Lili Gan

    Full Text Available Preventive anti-HPV vaccines are effective against HPV infection but not against existing HPV-associated diseases, including cervical cancer and other malignant diseases. Therefore, the development of therapeutic vaccines is urgently needed. To improve anti-tumor effects of therapeutic vaccine, we fused cytotoxic T-lymphocyte antigen 4 (CTLA-4 with HPV16 E7 and E6 as a fusion therapeutic DNA vaccine (pCTLA4-E7E6. pCTLA4-E7E6 induced significantly higher anti-E7E6 specific antibodies and relatively stronger specific CTL responses than the nonfusion DNA vaccine pE7E6 in C57BL/6 mice bearing with TC-1 tumors. pCTLA4-E7E6 showed relatively stronger anti-tumor effects than pE7E6 in therapeutic immunization. These results suggest that fusing CTLA-4 with E7E6 is a useful strategy to develop therapeutic HPV DNA vaccines. In addition, fusing the C-terminal of E7 with the N-terminal of E6 impaired the functions of both E7 and E6.

  9. DNA extraction from crayfish exoskeleton

    National Research Council Canada - National Science Library

    Li, Yanhe; Wang, Weimin; Liu, Xiaolian; Luo, Wei; Zhang, Jie; Gul, Yasmeen

    2011-01-01

    .... However, it is difficult to extract DNA from them. This study was intended to investigate CE as a DNA source and design an easy and efficient DNA extraction protocol for polymerase chain reactions...

  10. Sensitivity, specificity, and clinical value of human papillomavirus (HPV) E6/E7 mRNA assay as a triage test for cervical cytology and HPV DNA test.

    Science.gov (United States)

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Giorgi Rossi, Paolo

    2011-07-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  11. Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer

    NARCIS (Netherlands)

    M.F.D. Baay (Marc); J. Koudstaal; H. Hollema; J.M. Duk; M.P.M. Burger; W.G.V. Quint (Wim); E. Stolz (Ernst); P. Herbrink (Paul)

    1997-01-01

    textabstractThe prognostic value of detection of human papillomavirus (HPV) type 16 DNA in histologically cancer free lymph nodes was assessed in left obturator lymph nodes from cervical cancer patients with HPV-16 positive primary tumours. HPV-16 DNA was detected by po

  12. Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer

    NARCIS (Netherlands)

    Baay, MFD; Koudstaal, J; Hollema, H; Duk, JM; Burger, MPM; Quint, WGV; Stolz, E; Herbrink, P

    1997-01-01

    The prognostic value of detection of human papillomavirus (HPV) type 16 DNA in histologically cancer free lymph nodes was assessed in left obturator lymph nodes from cervical cancer patients with HPV-16 positive primary tumours. HPV-16 DNA was detected by polymerase chain reaction in 12 of 35 patien

  13. Detection of HPV-16 DNA by PCR in histologically cancer free lymph nodes from patients with cervical cancer

    NARCIS (Netherlands)

    M.F.D. Baay (Marc); J. Koudstaal; H. Hollema; J.M. Duk; M.P.M. Burger; W.G.V. Quint (Wim); E. Stolz (Ernst); P. Herbrink (Paul)

    1997-01-01

    textabstractThe prognostic value of detection of human papillomavirus (HPV) type 16 DNA in histologically cancer free lymph nodes was assessed in left obturator lymph nodes from cervical cancer patients with HPV-16 positive primary tumours. HPV-16 DNA was detected by po

  14. HPV DNA target hybridization concentrations studies using interdigitated electrodes (IDE) for early detection of cervical cancer

    Science.gov (United States)

    Noriani, C.; Hashim, U.; Azizah, N.; Nadzirah, Sh.; Arshad, M. K. Md; Ruslinda, A. R.; Gopinath, Subash C. B.

    2017-03-01

    Human Papillomaviruses (HPV) is the major cause of cervical cancer. HPV 16 and HPV 18 are the two types of HPV are the most HPV-associated cancers and responsible as a high-risk HPV. Cervical cancer took about 70 percent of all cases due to HPV infections. Cervical cancer mostly growth on a woman's cervix and its was developed slowly as cancer. TiO2 particles give better performance and low cost of the biosensor. The used of 3-aminopropyl triethoxysilane (APTES) will be more efficient for DNA nanochip. APTES used as absorption reaction to immobilize organic biomolecules on the inorganic surface. Furthermore, APTES provide better functionalization of the adsorption mechanism on IDE. The surface functionalized for immobilizing the DNA, which is the combination of the DNA probe and the HPV target produces high sensitivity and speed detection of the IDE. The Current-Voltage (IV) characteristic proved the sensitivity of the DNA nanochip increase as the concentration varied from 0% concentration to 24% of APTES concentration.

  15. Comparison of the clinical performance of an HPV mRNA test and an HPV DNA test in triage of atypical squamous cells of undetermined significance (ASC-US)

    DEFF Research Database (Denmark)

    Waldstrom, M; Ornskov, D

    2012-01-01

    The effect of triaging women with atypical squamous cells of undetermined significance (ASC-US) with human papillomavirus (HPV) DNA testing has been well documented. New tests detecting HPV E6/E7 mRNA are emerging, claiming to be more specific for detecting high-grade disease. We evaluated...... the clinical performance of two HPV tests: the Linear Array HPV genotyping test (LA) detecting HPV DNA from 37 oncogenic and non-oncogenic HPV types and the Aptima HPV assay detecting E6/E7 mRNA from 14 oncogenic HPV types....

  16. HPV E6/E7 mRNA versus HPV DNA biomarker in cervical cancer screening of a group of Macedonian women.

    Science.gov (United States)

    Duvlis, Sotirija; Popovska-Jankovic, Katerina; Arsova, Zorica Sarafinovska; Memeti, Shaban; Popeska, Zaneta; Plaseska-Karanfilska, Dijana

    2015-09-01

    High risk types of human papillomaviruses E6/E7 oncogenes and their association with tumor suppressor genes products are the key factors of cervical carcinogenesis. This study proposed them as specific markers for cervical dysplasia screening. The aim of the study is to compare the clinical and prognostic significance of HPV E6/E7 mRNA as an early biomarker versus HPV DNA detection and cytology in triage of woman for cervical cancer. The study group consists of 413 women: 258 NILM, 26 ASC-US, 81 LSIL, 41 HSIL, and 7 unsatisfactory cytology. HPV4AACE screening, real-time multiplex PCR and MY09/11 consensus PCR primers methods were used for the HPV DNA detection. The real-time multiplex nucleic acid sequence-based assay (NucliSENS EasyQ HPV assay) was used for HPV E6/E7 mRNA detection of the five most common high risk HPV types in cervical cancer (16, 18, 31, 33, and 45). The results show that HPV E6/E7 mRNA testing had a higher specificity 50% (95% CI 32-67) and positive predictive value (PPV) 62% (95% CI 46-76) for CIN2+ compared to HPV DNA testing that had specificity of 18% (95% CI 7-37) and PPV 52% (95% CI 39-76) respectively. The higher specificity and PPV of HPV E6/E7 mRNA testing are valuable in predicting insignificant HPV DNA infection among cases with borderline cytological finding. It can help in avoiding aggressive procedures (biopsies and over-referral of transient HPV infections) as well as lowering patient's anxiety and follow up period. © 2015 Wiley Periodicals, Inc.

  17. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil®.

    Science.gov (United States)

    Lee, Sin Hang

    2014-03-01

    Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results.

  18. Value and feasibility of HPV DNA test in cervical scraping smears.

    Science.gov (United States)

    Wu, Sufang; Chen, Gang; Wang, Wei; Xu, Qian; Gu, Hainian; Lu, Yunping; Zhou, Liping; Du, Juan; Li, Fujun; Liao, Guoning; Ma, Ding

    2005-01-01

    To investigate the reliability and feasibility of human papillomavirus (HPV) DNA test in cervical scraping smears with polymerase chain reaction (PCR), 131 cases of cervical scraping specimens were collected, and the positive rates and accuracy of HPV infection were determined in normal subjects and cervical cancer patients. GP5+/GP6+ and E7 primer pairs designed for detecting HPV L1 and HPV type 16 E7 were tested in this study. Our results showed that positive rates of HPV DNA in normal population and cervical cancer patients were 32.99% and 73.53% respectively and there was significant difference between them (P 0.05). However the detection rates in cervical cancer patients were 38.24% and 67.65% for the two markers, with a significant difference found between them (P scraping smears was feasible. GP5+/GP6+ primer pairs may be a useful probe to screen HPV infection in normal population, but they are not sensitive enough in cervical cancer patients. It is suggested that high risk type HPV DNA test was very useful in population with high risk of cervical cancer.

  19. Clustering self-organizing maps (SOM) method for human papillomavirus (HPV) DNA as the main cause of cervical cancer disease

    Science.gov (United States)

    Bustamam, A.; Aldila, D.; Fatimah, Arimbi, M. D.

    2017-07-01

    One of the most widely used clustering method, since it has advantage on its robustness, is Self-Organizing Maps (SOM) method. This paper discusses the application of SOM method on Human Papillomavirus (HPV) DNA which is the main cause of cervical cancer disease, the most dangerous cancer in developing countries. We use 18 types of HPV DNA-based on the newest complete genome. By using open-source-based program R, clustering process can separate 18 types of HPV into two different clusters. There are two types of HPV in the first cluster while 16 others in the second cluster. The analyzing result of 18 types HPV based on the malignancy of the virus (the difficultness to cure). Two of HPV types the first cluster can be classified as tame HPV, while 16 others in the second cluster are classified as vicious HPV.

  20. Validation study of HPV DNA detection from stained FNA smears by polymerase chain reaction

    DEFF Research Database (Denmark)

    Channir, Hani Ibrahim; Grønhøj Larsen, Christian; Ahlborn, Lise Barlebo;

    2016-01-01

    BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC) often presents with cystic cervical metastasis and a small primary tumor localized in the palatine tonsils or base of the tongue, which is diagnostically challenging. Testing for HPV DNA in fine......-needle aspiration (FNA) smears from metastases may facilitate a targeted diagnostic workup for identifying the primary tumor. This study was designed to assess the ability to detect HPV DNA in FNA smears with polymerase chain reaction (PCR). METHODS: May-Grünvald-Giemsa (MGG)-stained FNA smears from metastases...... and corresponding surgical specimens were collected from 71 patients with known HPV-positive OPSCC, 12 patients with oral squamous cell carcinoma (OSCC), 20 patients with branchial cleft cysts, and 20 patients with Warthin tumors. Thirty-eight patients with OPSCC and 7 patients with OSCC had FNA smears available...

  1. Hypoxia inducible factor-1 alpha expression is increased in infected positive HPV16 DNA oral squamous cell carcinoma and positively associated with HPV16 E7 oncoprotein

    Directory of Open Access Journals (Sweden)

    Di Fede Olga

    2011-10-01

    Full Text Available Abstract Background There is increasing evidence for the role of High Risk (HR Human PapillomaVirus (HPV in the pathogenesis of Oral Squamous Cell Carcinoma (OSCC. The E6 and E7 oncogenes from HR HPVs are responsible for the deregulation of p53 and pRB proteins involved in cell cycle and apoptotic pathways. In cell lines experiments, the HPV E7 protein seems to be able to enhance Hypoxia Inducible Factor-1 alpha (HIF-1α activity, normally involved in the response to hypoxia and able to enhance angiogenesis. Results We studied tumor specimens from 62 OSCC; a higher prevalence of tumors in TNM stage II and also in pT2 class between OSCC infected positive HPV16 DNA than non-infected ones was observed. HIF-1α positivity was detected throughout the analysed fields, not associated with areas of necrosis and also observed in cells immediately adjacent to blood vessels. A significant increase in mean values of the HIF-1α labeling indexes was observed for pT1-T2, as well for stage I-II, in the infected positive HPV16 DNA tumors than non-infected ones. HIF-1α and HPV16 E7 labeling indexes showed a significantly positive correlation which suggested a positive association between HPV16 E7 and HIF-1α expression. Conclusions In our specimens HIF-1α immunoreactivity hints for an O2-independent regulatory mechanism in infected positive HPV16 DNA tumors, especially for pT1-T2 and stage I-II tumors, suggesting a very early involvement in the development of HPV-induced OSCC. HIF-1α and HPV16 E7 labeling indexes suggest also a positive association between the two proteins in infected positive HPV16 DNA OSCC.

  2. Prevalence of HPV DNA in cervical lesions in patients from Ecuador and Japan.

    Science.gov (United States)

    Paez, C; Konno, R; Yaegashi, N; Matsunaga, G; Araujo, I; Corral, F; Sato, S; Yajima, A

    1996-11-01

    Cervical cancer is about 6 times more frequent in Ecuador than in Japan. We investigated the association between infection by the human papillomavirus (HPV) and the genesis of cervical cancer in specimens of lesions of the cervical epithelium obtained from patients in Ecuador and Japan. We also examined the results of HPV DNA detection and typing by the polymerase chain reaction (PCR) performed under the same technical conditions in areas with differing rates of cervical cancer. Purified tissue DNA from paraffin-embedded samples was amplified by PCR with universal and type-specific primers. HPV DNA was detected in 8 (20%) of 40 normal cervical epithelial samples from Ecuadorian patients, 19 (45%) of 42 low-grade squamous intraepithelial lesions (LSIL), 16 (50%) of 32 high-grade squamous intraepithelial lesions (HSIL), and 38 (81%) of 47 invasive squamous cell carcinomas (SCC) compared with 3 (10%) of 30 normal cervical specimens from Japanese patients, 107 (51%) of 210 HSILs, and 45 (71%) of 63 SCCs. The prevalence of HPV types 16 and 18 rose significantly with increasing histological grade (p poverty and underdevelopment may influence the prevalence of HPV infection and the sequence of events after HPV infection culminating in cervical cancer. These factors may help to explain the differing geographic distribution of this disease.

  3. Detection of Human Papillomavirus DNA by AffiProbe HPV-DNA Test Kit in Cervical Scrapes or Biopsies-Histopathologic Correlates

    OpenAIRE

    Pekka Nieminen; Tarja Jalava; Arja Kallio; Marjut Ranki; Jorma Paavonen

    1994-01-01

    Objective: The aim of this study was to evaluate and compare the efficacy of punch biopsies and cervical scrapes in the detection of human papillomavirus (HPV) DNA from the cervix and compare the results with the histopathologic diagnosis. Methods: The specimens were collected simultaneously, and HPV DNA was detected using a liquid hybridization test. Results: Biopsies and scrapes were equally efficient, but each detected only two-thirds of all HPV-DNA-positive patients. Thus, the positivity ...

  4. Prevalence of human papillomavirus (HPV) and HPV-16 genotyping by real-time PCR in patients with several cervical pathologies.

    Science.gov (United States)

    Dinc, Bedia; Rota, Seyyal; Onan, Anil; Bozdayi, Gulendam; Taskiran, Cagatay; Biri, Aydan; Güner, Haldun

    2010-01-01

    this study was planned to evaluate the prevalence of HPV (excepting type 16) and HPV 16 by real-time PCR in colposcopy patients and to interprete the results with age, age of first sexual intercourse (FSI), parity and Pap smear results. one hundred and two colposcopy patients (50 and 52 of the patients were classified as colposcopy positive and negative, respectively) applying to Gynecology clinic were included. HPV (excepting type 16) and HPV 16 were detected by realtime PCR using the L1 region. Real-time nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV and HPV 16 DNA specific probe after HPV DNA extraction by phenol chloroform isoamylalcohol. HPV (excepting type 16) and HPV 16 were positive in 12% and 18% of the colposcopy positive patients respectively. HPV (excepting type 16) and HPV 16 were positive in 5.7% and 3.8% of the colposcopy negative patients, respectively. there was a statistically significant difference between colposcopy positive and colposcopy negative patients comparing HPV 16 with total HPV positivity (p = 0.021 for type 16 and p = 0.010 for total HPV) but there was not a statistically significant difference between colposcopy positive and colposcopy negative patients when we compared HPV (excepting type 16) positivity (p = 0.314). In conclusion, HPV detection and typing may be helpful for cervical cancer screening and prevention.

  5. Prevalence of human papillomavirus (HPV and HPV-16 genotyping by real-time PCR in patients with several cervical pathologies

    Directory of Open Access Journals (Sweden)

    Bedia Dinc

    Full Text Available PURPOSE: this study was planned to evaluate the prevalence of HPV (excepting type 16 and HPV 16 by real-time PCR in colposcopy patients and to interprete the results with age, age of first sexual intercourse (FSI, parity and Pap smear results. METHODS: one hundred and two colposcopy patients (50 and 52 of the patients were classified as colposcopy positive and negative, respectively applying to Gynecology clinic were included. HPV (excepting type 16 and HPV 16 were detected by realtime PCR using the L1 region. Real-time nested amplifications of MY09/11 products were done by GP5+/GP6+ primers and Cyanine-5 labeled HPV and HPV 16 DNA specific probe after HPV DNA extraction by phenol chloroform isoamylalcohol. RESULTS: HPV (excepting type 16 and HPV 16 were positive in 12% and 18% of the colposcopy positive patients respectively. HPV (excepting type 16 and HPV 16 were positive in 5.7% and 3.8% of the colposcopy negative patients, respectively. CONCLUSION: there was a statistically significant difference between colposcopy positive and colposcopy negative patients comparing HPV 16 with total HPV positivity (p = 0.021 for type 16 and p = 0.010 for total HPV but there was not a statistically significant difference between colposcopy positive and colposcopy negative patients when we compared HPV (excepting type 16 positivity (p = 0.314. In conclusion, HPV detection and typing may be helpful for cervical cancer screening and prevention.

  6. Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays

    DEFF Research Database (Denmark)

    Iftner, Thomas; Germ, Liesje; Swoyer, Ryan

    2009-01-01

    Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different...... methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay...... (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons...

  7. Event extraction for DNA methylation

    Directory of Open Access Journals (Sweden)

    Ohta Tomoko

    2011-10-01

    Full Text Available Abstract Background We consider the task of automatically extracting DNA methylation events from the biomedical domain literature. DNA methylation is a key mechanism of epigenetic control of gene expression and implicated in many cancers, but there has been little study of automatic information extraction for DNA methylation. Results We present an annotation scheme for DNA methylation following the representation of the BioNLP shared task on event extraction, select a set of 200 abstracts including a representative sample of all PubMed citations relevant to DNA methylation, and introduce manual annotation for this corpus marking nearly 3000 gene/protein mentions and 1500 DNA methylation and demethylation events. We retrain a state-of-the-art event extraction system on the corpus and find that automatic extraction of DNA methylation events, the methylated genes, and their methylation sites can be performed at 78% precision and 76% recall. Conclusions Our results demonstrate that reliable extraction methods for DNA methylation events can be created through corpus annotation and straightforward retraining of a general event extraction system. The introduced resources are freely available for use in research from the GENIA project homepage http://www-tsujii.is.s.u-tokyo.ac.jp/GENIA.

  8. HPV-DNA testing and Ki-67 immunocytochemistry in liquid based cervical cytology in prostitute women

    Directory of Open Access Journals (Sweden)

    Esin ATİK

    2006-09-01

    Full Text Available HPV causes several changes in the function of host genes, and these interactions cause deregulation of the cell cycle manifested by abnormal expression of cell cycle associated proteins, such as Ki-67. The detection of Ki-67 can play a role in screening and diagnosis of HPV infection with risk of progression towards dysplasia and carcinoma. To show this relation in prostitute women, cervical cells were collected in the PapSpin Collection Fluid. A starting volume of 1000 µl for each sample, and a 200 µl cell suspension were used to prepare each sample for thin layer liquid based cytology and then they were stained by Papanicolaou method. The cytological results were classified according to the Bethesda 2001 system. From the remaining cell suspension of 800 µl, a 400 µl sample was used for HPV-DNA detection by PCR, a 50 µl alliquot was used to make thin layer preparations for immunocytochemistry. Single antigen staining was performed with Ki-67 protein. Cells were considered immunopositive if the nuclei were stained. All cells in one high power field (x400 were counted, and the fraction of immunopositive cells on the slide was calculated. This fraction was expressed as the number of positive cells per 1000 cells to facilitate comparisons of differential cell counts. HPV types 6 and 32 in the study, and HPV types 6 and 51 in the control group were detected. The mean Ki-67 values were 2.7±1.2 and 3.6±4.1 in HPV positive and negative cases respectively. There was a positive correlation only with nuclear changes and HPV positivity (x2=28.8, p<0.001. There was not any significant correlation between HPV or Ki-67 and leukocytosis. An association with HPV and contraception, smoking, and concurrent genital infection was not found. The prevalence of HPV types in different geographical locations and races may indicate different etiologies of cervical cancer. Our results suggest that Ki-67 immunocytochemistry is not useful as a surrogate marker for

  9. Simultaneous in situ hybridization for DNA and RNA reveals the presence of HPV in the majority of cervical cancer cells.

    Science.gov (United States)

    D'Amato, L; Pilotti, S; Longoni, A; Donghi, R; Rilke, F

    1992-02-01

    Thirteen cases of invasive squamous cell carcinoma of the uterine cervix containing HPV types 16 or 18 DNA sequences, as detected by Southern blot analysis, were investigated by in situ hybridization on routine paraffin sections, using 35S nick-translated DNA probes. Simultaneous in situ hybridization for DNA and RNA showed that in ten out of 13 cases (77%) the percentage of tumor cells containing HPV 16 or 18 varied from 75 to 100%. In one case, harboring both in situ and invasive carcinoma, the same type of HPV DNA was detected in both components. This finding suggests that neoplastic cells retained the viral genome during progression to invasiveness.

  10. Liquid-based cytology and HPV DNA testing using intra-anal specimens from HIV-negative women with and without genital HPV-induced lesions.

    Science.gov (United States)

    Eleutério, José; Benício, Givanildo Carneiro; Giraldo, Paulo César; Gonçalves, Ana Katherine Silveira; Eleutério, Renata Mírian Nunes; Oliveira, Denise Nunes; Jacyntho, Cláudia

    2015-05-01

    Screening for anal cancer using cytology has not been considered in immunocompetent women. The aim of this study was to identify cytological atypia and human papillomavirus (HPV) DNA in intra-anal specimens from human immunodeficiency virus (HIV)-negative women with and without genital HPV lesions. This study was a cross-sectional analysis of 142 women who were negative for the HIV: 80 with genital lesions that were associated with HPV and 62 without HPV-induced lesions. The women were evaluated at the Federal University of Ceará from October 2011 to June 2012. The statistical analysis included the Fisher exact test and the odds ratio (CI 95%). Atypical anal cytology was observed in 24 (29.3%) patients in the study group and in 11 (17.8%) patients in the control group. In cases with at least two sites of HPV-associated lesions, 12 (41.4%) presented atypical cytology (P = 0.0220; OR = 2.7621, 1.1579-6.5889). When the practice of anal sex was evaluated, atypical cytology was observed in 22/43 (34%) [P = 0.0214; OR = 2.519, 1.146-5.534]. HPV DNA was detected in 17/27 (63%) cases with at least two sites of lesions (P = 0.0293, OR = 2.4855, 1.0960-5.6367). In the 33 cases who presented positive HPV DNA test results, the liquid-based cytology results were atypical (P = 0.0212, OR = 2.8, 1.1665-6.7208). Based on the results, liquid-based cytology may be used to detect intra-anal lesions, especially among women who have a history of anal intercourse or who have genital HPV-associated lesions at multiple sites. © 2014 Wiley Periodicals, Inc.

  11. [Cervix uteri lesions and human papiloma virus infection (HPV): detection and characterization of DNA/HPV using PCR (polymerase chain reaction].

    Science.gov (United States)

    Serra, H; Pista, A; Figueiredo, P; Urbano, A; Avilez, F; De Oliveira, C F

    2000-01-01

    The prevalence of human papillomavirus (HPV) genotypes was estimated by the polymerase chain reaction (PCR), in archival paraffin was embedded tissues. The case group consisted of 84 women aged 21-67 years (mean, 40 years) who were referred to the Department of Gynaecology (Oncology Centre, Coimbra) with citopathologically abnormal smears. This group was selected from a population of women who had undergone a screening programme (1990/94) in Central Region of Portugal. All these patients (n = 84) had a colposcopic directed cervical biopsy. HPV detection and typing was performed by the PCR method in the Department of Virology (National Health Care Institute, Lisbon). The prevalence of DNA/HPV found, concerning all epithelial cervical lesions studied and classified as squamous intra-epithelial lesions (SIL) and cervical cancer was 97.8%. On the basis of the data presented in this study, it was estimated that there was a statistically significant prevalence of low risk HPV types (HPV 6/11) in low grade SIL, 83.3%, and a statistically significant prevalence of high risk HPV types (HPV 16,18,31,33,51) in high grade SIL, 58.4%, as well as cervical cancer lesions in 100%. We conclude that there was a statistically significant difference between women with low and high grade SIL for HPV infection, with low and high risk HPV types, respectively. The risk factors for cervical cancer investigated (age at first sexual intercourse, multiple sexual partners, parity, use of oral contraceptives) were not associated to statistically significant differences concerning low grade SIL and high grade SIL. The clinical and therapeutic procedures were evaluated for the same five years (1990/94). It may be concluded that there would be no significant difference in clinical procedure for high grade lesions and cervical cancer, in which the treatment had been frequently radical (cone biopsies, simple or radical hysterectomy) and in which the HPV infection persisted frequently and was

  12. DNA methylation analysis in self-sampled brush material as a triage test in hrHPV-positive women

    NARCIS (Netherlands)

    Boers, A.; Bosgraaf, R. P.; van Leeuwen, R. W.; Schuuring, E.; Heideman, D. A. M.; Massuger, L. F. A. G.; Verhoef, V. M. J.; Bulten, J.; Melchers, W. J. G.; van der Zee, A. G. J.; Bekkers, R. L. M.; Wisman, G. B. A.

    2014-01-01

    Background: Primary high-risk human papillomavirus (hrHPV) testing in cervical cancer screening shows relatively low specificity, which makes triage testing necessary. In this study, DNA methylation analysis was compared with cytology for triage testing in hrHPV-positive women. Moreover, feasibility

  13. DNA methylation analysis in self-sampled brush material as a triage test in hrHPV-positive women

    NARCIS (Netherlands)

    Boers, A.; Bosgraaf, R.P.; Leeuwen, R.W. van; Schuuring, E.; Heideman, D.A.; Massuger, L.F.; Verhoef, V.M.; Bulten, J.; Melchers, W.J.; Zee, A.G. van der; Bekkers, R.L.; Wisman, G.B.

    2014-01-01

    BACKGROUND: Primary high-risk human papillomavirus (hrHPV) testing in cervical cancer screening shows relatively low specificity, which makes triage testing necessary. In this study, DNA methylation analysis was compared with cytology for triage testing in hrHPV-positive women. Moreover, feasibility

  14. DNA methylation analysis in self-sampled brush material as a triage test in hrHPV-positive women

    NARCIS (Netherlands)

    Boers, A.; Bosgraaf, R. P.; van Leeuwen, R. W.; Schuuring, E.; Heideman, D. A. M.; Massuger, L. F. A. G.; Verhoef, V. M. J.; Bulten, J.; Melchers, W. J. G.; van der Zee, A. G. J.; Bekkers, R. L. M.; Wisman, G. B. A.

    2014-01-01

    Background: Primary high-risk human papillomavirus (hrHPV) testing in cervical cancer screening shows relatively low specificity, which makes triage testing necessary. In this study, DNA methylation analysis was compared with cytology for triage testing in hrHPV-positive women. Moreover, feasibility

  15. The clinical utility of HPV DNA testing in cervical cancer screening strategies.

    Science.gov (United States)

    Bhatla, Neerja; Moda, Nidhi

    2009-09-01

    Cervical cancer continues to be the commonest cause of death among women in developing countries, largely due to the failure to the inability to sustain effective cytology-based screening programs. While this burden may come down following implementation of the human papillomavirus (HPV) vaccine, screening will still be required. HPV DNA testing is a promising new technology for cervical cancer prevention and is the most reproducible of all cervical cancer screening tests. Presently, the two assays most widely used for the detection of genital types are the polymerase chain reaction (PCR) and Hybrid Capture 2 assays (hc2). Rapid, affordable tests are expected to be available soon. HPV DNA testing can be used in a variety of clinical scenarios that include primary screening in women older than 30 yr; as an adjunctive test to cytology; in the triage of women with an equivocal cytologic report, e.g., ASC-US; or for follow-up post-treatment for cervical intraepithelial neoplasia (CIN). HPV DNA testing can also be performed on self-collected samples, which allows screening in remote areas and also in women who refuse gynecologic examination.

  16. [Health technology assessment report: HPV DNA based primary screening for cervical cancer precursors].

    Science.gov (United States)

    Ronco, Guglielmo; Biggeri, Annibale; Confortini, Massimo; Naldoni, Carlo; Segnan, Nereo; Sideri, Mario; Zappa, Marco; Zorzi, Manuel; Calvia, Maria; Accetta, Gabriele; Giordano, Livia; Cogo, Carla; Carozzi, Francesca; Gillio Tos, Anna; Arbyn, Marc; Mejier, Chris J L M; Snijders, Peter J F; Cuzick, Jack; Giorgi Rossi, Paolo

    2012-01-01

    OBJECTIVE OF THE PROJECT: The introduction of the HPV test as a primary screening test will cause important changes in the screening system based on cytology. The purposes of this report are: to define the best screening policies with HPV-based screening on the basis of the resulting efficacy and of undesired effects; comparing them to cytology-based screening; to identify their best conditions of application; to evaluate economic cost, feasibility and impact on the organisation of services of such policy in the Italian situation. This report contains a section on efficacy and undesired effects based on a systematic review of literature conducted in strict coordination with the preparation of a supplement to the European Guidelines for quality assurance in cervical cancer screening. This chapter corresponds to a preliminary version of the chapter of the European Guidelines on primary screening with HPV. The sections on costs, impact on organisation, and social, ethical and legal impact reflect the Italian situation; they are based on a review of the available Italian data (including unpublished data, mainly from on-going pilot projects) and on a structured analysis of what will result if the proposed protocol is applied to the Italian situation. Efficacy and undesired effects. There is clear scientific evidence that a screening based on validated tests for the DNA of oncogenic HPV as primary test and applying an appropriate protocol is more effective than screening based on cytology in preventing invasive cancers of the uterine cervix. In addition, it entails a limited--if any--increase of the undesired effects both in terms of unneeded referral to diagnostic work-up and in terms of over-diagnosis and consequent overtreatment of spontaneously regressive lesions. The crucial elements of such protocol are the followings: HPV-positive women are not to be directly referred to colposcopy, but the use of triage systems is essential. The currently recommendable method is

  17. Human papillomavirus (HPV detected in restored plasma DNA from women diagnosed with pre-invasive lesions and invasive cervical cancer

    Directory of Open Access Journals (Sweden)

    Yazmín Rocío Arias

    2010-06-01

    Full Text Available Objective: To improve the sensitivity of Human Papillomavirus (HPV detection in plasma from high-grade cervical neoplasia patients (CIN III and cervical cancer (CC evaluating any likely correlation with disease stage. Method: We subjected plasma DNA isolates from 112 patients (CIN and ICC to a pre-PCR restoration treatment to improve detection sensitivity. HPV-specific sequences were detected by conventional PCR both in cervical scrapes and plasma DNA obtained from each patient. For every single DNA sample, both non-restored and restored isolates were PCR analyzed. Results: We detected HPV in plasma DNA isolates with significantly higher efficiency on restored plasma-DNA as compared to each non-restored equivalent, still maintaining close correlation with the clinical stage of the cases. By analyzing plasma-DNA isolates we could classify as HPV positive >50.0% of the cases that were previously known to be positive from the cervical scrape based assay. Interestingly, 100% of the cases in which subtype HPV18 was detected in cervical scrapes were also positive in plasma DNA. Conclusions: Restoration of plasma DNA from cervical cancer patients allows a more sensitive PCR-based HPV detection, maintaining the correlation to disease stage traditionally observed.

  18. Human papillomavirus (HPV detected in restored plasma DNA from women diagnosed with pre-invasive lesions and invasive cervical cancer

    Directory of Open Access Journals (Sweden)

    Edward Fabián Carrillo

    2010-06-01

    Full Text Available Objective: To improve the sensitivity of Human Papillomavirus (HPV detection in plasma from high-grade cervical neoplasia patients (CIN III and cervical cancer (CC evaluating any likely correlation with disease stage.Method: We subjected plasma DNA isolates from 112 patients (CIN and ICC to a pre-PCR restoration treatment to improve detection sensitivity. HPV-specific sequences were detected by conventional PCR both in cervical scrapes and plasma DNA obtained from each patient. For every single DNA sample, both non-restored and restored isolates were PCR analyzed.Results: We detected HPV in plasma DNA isolates with significantly higher efficiency on restored plasma-DNA as compared to each non-restored equivalent, still maintaining close correlation with the clinical stage of the cases. By analyzing plasma-DNA isolates we could classify as HPV positive >50.0% of the cases that were previously known to be positive from the cervical scrape based assay. Interestingly, 100% of the cases in which subtype HPV18 was detected in cervical scrapes were also positive in plasma DNA.Conclusions: Restoration of plasma DNA from cervical cancer patients allows a more sensitive PCR-based HPV detection, maintaining the correlation to disease stage traditionally observed.

  19. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus;

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible ...

  20. Automated Extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...... the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles....

  1. Comparison of five protocols to extract DNA from paraffin-embedded tissues for the detection of human papillomavirus.

    Science.gov (United States)

    Alvarez-Aldana, Adalucy; Martínez, José William; Sepúlveda-Arias, Juan C

    2015-02-01

    Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable source of DNA with which to perform large retrospective studies on the epidemiology of HPV infection. Five different DNA extraction protocols were carried out to evaluate the DNA obtained from FFPE samples with polymerase chain reaction (PCR) using two primer sets to amplify a constitutive human gene, β-globin, and two primer sets to detect the L1 and E6 HPV genes. From the five DNA extraction protocols evaluated, the best results were obtained with protocol A, corresponding to a crude extract from the sample. With the procedures described herein, we were able to amplify DNA extracted from archival paraffin blocks stored for six years. However, the amplification products were more efficiently obtained with primers that amplified shorter fragments. This result indicates that a major factor limiting the extraction process in these samples is DNA fragmentation, a factor that will naturally vary between the different specimens evaluated. Also, depending upon the extraction method, PCR amplification of a human gene does not necessarily guarantee the successful extraction of viral DNA. In conclusion, different DNA and HPV detection methods can significantly influence the results. Therefore, the DNA extraction methods and primers used for DNA amplification in fixed tissues need to be chosen carefully, depending on the specific requirements of the study being carried out.

  2. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible...... to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range....

  3. Repeated extraction of DNA from FTA cards

    OpenAIRE

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus; Frank-Hansen, Rune; Hansen, Anders Johannes; Morling, Niels

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range.

  4. Repeated extraction of DNA from FTA cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Ferrero, Laura; Børsting, Claus

    2011-01-01

    Extraction of DNA using magnetic bead based techniques on automated DNA extraction instruments provides a fast, reliable and reproducible method for DNA extraction from various matrices. However, the yield of extracted DNA from FTA-cards is typically low. Here, we demonstrate that it is possible...... to repeatedly extract DNA from the processed FTA-disk. The method increases the yield from the nanogram range to the microgram range....

  5. Sensitivity, Specificity, and Clinical Value of Human Papillomavirus (HPV) E6/E7 mRNA Assay as a Triage Test for Cervical Cytology and HPV DNA Test ▿

    Science.gov (United States)

    Benevolo, Maria; Vocaturo, Amina; Caraceni, Donatella; French, Deborah; Rosini, Sandra; Zappacosta, Roberta; Terrenato, Irene; Ciccocioppo, Lucia; Frega, Antonio; Rossi, Paolo Giorgi

    2011-01-01

    There is evidence that testing for human papillomavirus (HPV) E6/E7 mRNA is more specific than testing for HPV DNA. A retrospective study was carried out to evaluate the performance of the PreTect HPV-Proofer E6/E7 mRNA assay (Norchip) as a triage test for cytology and HPV DNA testing. This study analyzed 1,201 women, 688 of whom had a colposcopy follow-up and 195 of whom had histology-confirmed high-grade intraepithelial neoplasia or worse (CIN2+). The proportion of positive results and the sensitivity and specificity for CIN2+ were determined for HPV mRNA in comparison to HPV DNA and cytology. All data were adjusted for follow-up completeness. Stratified by cytological grades, the HPV mRNA sensitivity was 83% (95% confidence interval [CI] = 63 to 94%) in ASC-US (atypical squamous cells of undetermined significance), 62% (95% CI = 47 to 75%) in L-SIL (low-grade squamous intraepithelial lesion), and 67% (95% CI = 57 to 76%) in H-SIL (high-grade squamous intraepithelial lesion). The corresponding figures were 99, 91, and 96%, respectively, for HPV DNA. The specificities were 82, 76, and 45%, respectively, for HPV mRNA and 29, 13, and 4%, respectively, for HPV DNA. Used as a triage test for ASC-US and L-SIL, mRNA reduced colposcopies by 79% (95% CI = 74 to 83%) and 69% (95% CI = 65 to 74%), respectively, while HPV DNA reduced colposcopies by 38% (95% CI = 32 to 44%) and by 15% (95% CI = 12 to 19%), respectively. As a HPV DNA positivity triage test, mRNA reduced colposcopies by 63% (95% CI = 60 to 66%), having 68% sensitivity (95% CI = 61 to 75%), whereas cytology at the ASC-US+ threshold reduced colposcopies by 23% (95% CI = 20 to 26%), showing 92% sensitivity (95% CI = 87 to 95%). In conclusion, PreTect HPV-Proofer mRNA can serve as a better triage test than HPV DNA to reduce colposcopy referral in both ASC-US and L-SIL. It is also more efficient than cytology for the triage of HPV DNA-positive women. Nevertheless, its low sensitivity demands a strict follow-up of

  6. DNA extraction from formalin-fixed material.

    Science.gov (United States)

    Campos, Paula F; Gilbert, Thomas M P

    2012-01-01

    The principal challenges facing PCR-based analyses of DNA extracted from formalin-fixed materials are fragmentation of the DNA and cross-linked protein-DNA complexes. Here, we present an efficient protocol to extract DNA from formalin-fixed or paraffin-embedded tissues (FFPE). In this protocol, protein-DNA cross-links are reversed using heat and alkali treatment, yielding significantly longer fragments and larger amounts of PCR-amplifiable DNA than standard DNA extraction protocols.

  7. Automated Extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas;

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing t...... the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles.......Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...

  8. Presence of high risk HPV DNA but indolent transcription of E6/E7 oncogenes in invasive ductal carcinoma of breast.

    Science.gov (United States)

    Wang, Depu; Fu, Ling; Shah, Walayat; Zhang, Jingwen; Yan, Yan; Ge, Xinhong; He, Jianjun; Wang, Yili; Li, Xu

    2016-12-01

    The causative role of high risk human papillomavirus (HR-HPV) in breast cancer development is controversial, though a number of reports have identified HR-HPV DNA in breast cancer specimens. Nevertheless, most studies to date have focused primarily on viral DNA rather than the viral transcription. The aim of this study was to investigate the presence of HR-HPV in breast cancer tissues at HPV DNA level and HPV oncogenes mRNA level by in situ hybridization (ISH). One hundred and forty six (146) cases of breast invasive ductal carcinoma(IDC) and 83 cases of benign breast lesions were included in the study. Type specific oligonucleotide probes were used for the DNA detection of HPV 16,18 and 58 by ISH. HR-HPV oncogenes mRNA was assayed by novel RNAscope HR-HPV HR7 assay ISH. p16 protein expression was evaluated by immunohistochemistry (IHC). HR-HPV 16,18 and 58 DNA were detected in 52 out of 146 (35.6%) IDC and in 3 out of 83 (3.6%) benign breast lesions by ISH. The HR-HPV mRNAs was detected only in a few specimens with strong HPV DNA positivity(4/25) in a few scattered cancer cells with very weak punctate nuclear and/or cytoplasmic staining. p16 over-expression did not correlate with the HPV DNA positive breast cancer samples(17/52 HPVDNA+ vs 28/94 HPV DNA-, p=0.731). HR-HPVs certainly exist in breast cancer tissue with less active transcription, which implies that the causal role of HPV in breast cancer development need further study. Copyright © 2016 Elsevier GmbH. All rights reserved.

  9. Two novel genital human papillomavirus (HPV) types, HPV68 and HPV70, related to the potentially oncogenic HPV39.

    OpenAIRE

    Longuet, M; Beaudenon, S; Orth, G

    1996-01-01

    The genomes of two novel human papillomavirus (HPV) types, HPV68 and HPV70, were cloned from a low-grade cervical intraepithelial neoplasia and a vulvar papilloma, respectively, and partially sequenced. Both types are related to HPV39, a potentially oncogenic virus. HPV68 and HPV70 were also detected in genital intraepithelial neoplasia from three patients and one patient, respectively. Comparison with sequence data in the literature indicates that the subgenomic ME180-HPV DNA fragment, clone...

  10. Automated DNA extraction from pollen in honey.

    Science.gov (United States)

    Guertler, Patrick; Eicheldinger, Adelina; Muschler, Paul; Goerlich, Ottmar; Busch, Ulrich

    2014-04-15

    In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. DNA Extraction and Primer Selection

    DEFF Research Database (Denmark)

    Karst, Søren Michael; Nielsen, Per Halkjær; Albertsen, Mads

    Talk regarding pitfalls in DNA extraction and 16S amplicon primer choice when performing community analysis of complex microbial communities. The talk was a part of Workshop 2 "Principles, Potential, and Limitations of Novel Molecular Methods in Water Engineering; from Amplicon Sequencing to -omics...

  12. DNA Extraction Techniques for Use in Education

    Science.gov (United States)

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  13. DNA Extraction Techniques for Use in Education

    Science.gov (United States)

    Hearn, R. P.; Arblaster, K. E.

    2010-01-01

    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  14. Use of AffiProbe HPV test kit for detection of human papillomavirus DNA in genital scrapes.

    OpenAIRE

    Ranki, M; Leinonen, A W; Jalava, T. (Tiina); Nieminen, P.; Soares, V R; Paavonen, J; Kallio, A

    1990-01-01

    The presence of human papillomavirus (HPV) DNA in cervical and vaginal scrapes was analyzed by the AffiProbe HPV test kit (Orion Corp., Orion Pharmaceutica, Helsinki, Finland), which is a 1-day solution hybridization test for HPV type 6/11, 16, or 18. The AffiProbe test was compared with a commercially available dot blot test (ViraPap and ViraType tests; Life Technologies Inc., Gaithersburg, Md.). The study group consisted of 178 patients seen in a gynecological outpatient clinic. Altogether,...

  15. Human Papillomavirus (HPV) Infection in Squamous Cell Carcinomas Arising From the Oropharynx: Detection of HPV DNA and p16 Immunohistochemistry as Diagnostic and Prognostic Indicators—A Pilot Study

    Energy Technology Data Exchange (ETDEWEB)

    Bussu, Francesco, E-mail: francesco.bussu.md@gmail.com [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Sali, Michela [Institute of Microbiology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Gallus, Roberto [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Petrone, Gianluigi; Zannoni, Gian Franco [Institute of Histopathology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Autorino, Rosa; Dinapoli, Nicola [Institute of Radiotherapy, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Santangelo, Rosaria [Institute of Microbiology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Vellone, Valerio Gaetano [Institute of Histopathology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Graziani, Cristina [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Miccichè, Francesco [Institute of Radiotherapy, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Almadori, Giovanni; Galli, Jacopo [Institute of Otolaryngology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Delogu, Giovanni; Sanguinetti, Maurizio [Institute of Microbiology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); Rindi, Guido [Institute of Histopathology, Università Cattolica del Sacro Cuore, Policlinico A. Gemelli, Roma (Italy); and others

    2014-08-01

    Purpose: Human papillomavirus (HPV) 16 infection is associated with oropharyngeal carcinogenesis and is likely the cause of the reported increase in disease incidence. We evaluated the prevalence of HPV infection and the reliability of different diagnostic tools using primary tumor samples from a cohort of 50 patients. Methods and Materials: Formalin-fixed paraffin-embedded (FFPE) tumor samples were collected from all 50 consecutive primary oropharyngeal SCC patients who were enrolled in the study; fresh tumor samples were available in 22 cases. NucliSENS EasyQ HPVv1 was used for RNA, and Digene Hybrid Capture-2(HC2) was used for DNA detection. p16 Expression was evaluated by immunohistochemistry in FPPE specimens. Results: Based on the DNA detection assay on FFPE samples, the frequency of high-risk HPV infection was 32%. The agreement rate between HPV RNA and HPV DNA detection in fresh samples was 100%. The agreement rate between p16 immunohistochemistry (IHC) and the detection of HPV DNA in the FFPE samples was fair but not excellent (κ = 0.618). HPV DNA detection was highly significant, as measured by disease-specific survival and determined using a Wilcoxon test (P=.001). p16 IHC also exhibited a prognostic value but with a lower statistical significance (P=.0475). The detection of HPV DNA, but not p16 IHC, was also significantly correlated with locoregional control (P=.0461). Conclusion: Diagnostic methods based on the detection of HPV nucleic acids appear to be more reliable and objective because they do not require reading by a trained histopathologist. Furthermore, the detection of HPV DNA exhibits an improved correlation with survival, and therefore appears definitely more reliable than p16 IHC for routine use in clinical practice.

  16. Comparison of Real-Time Multiplex Human Papillomavirus (HPV) PCR Assays with INNO-LiPA HPV Genotyping Extra Assay▿

    OpenAIRE

    Else, Elizabeth A.; Swoyer, Ryan; Zhang, Yuhua; Taddeo, Frank J.; Bryan, Janine T.; Lawson, John; Van Hyfte, Inez; Roberts, Christine C.

    2011-01-01

    Real-time type-specific multiplex human papillomavirus (HPV) PCR assays were developed to detect HPV DNA in samples collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). Additional multiplex (L1, E6, and E7 open reading frame [ORF]) or duplex (E6 and E7 ORF) HPV PCR assays were developed to detect high-risk HPV types, including HPV type 31 (HPV31), HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, and H...

  17. Evaluation of DNA and RNA extraction methods.

    Science.gov (United States)

    Edwin Shiaw, C S; Shiran, M S; Cheah, Y K; Tan, G C; Sabariah, A R

    2010-06-01

    This study was done to evaluate various DNA and RNA extractions from archival FFPE tissues. A total of 30 FFPE blocks from the years of 2004 to 2006 were assessed with each modified and adapted method. Extraction protocols evaluated include the modified enzymatic extraction method (Method A), Chelex-100 extraction method (Method B), heat-induced retrieval in alkaline solution extraction method (Methods C and D) and one commercial FFPE DNA Extraction kit (Qiagen, Crawley, UK). For RNA extraction, 2 extraction protocols were evaluated including the enzymatic extraction method (Method 1), and Chelex-100 RNA extraction method (Method 2). Results show that the modified enzymatic extraction method (Method A) is an efficient DNA extraction protocol, while for RNA extraction, the enzymatic method (Method 1) and the Chelex-100 RNA extraction method (Method 2) are equally efficient RNA extraction protocols.

  18. Comparison of Xpert(®) HPV and Hybrid Capture(®) 2 DNA Test™ for detection of high-risk HPV infection in cervical atypical squamous cells of undetermined significance.

    Science.gov (United States)

    Rabaan, Ali A; Taylor, Donald R; Dawamneh, Mohammed F; Al-Tawfiq, Jaffar A

    This study compares Xpert(®) HPV and Hybrid Capture(®) 2 High-Risk HPV DNA Test™ (hc2) for the detection of high-risk HPV infection in cervical smears. Papanicolaou smears with atypical squamous cells of undetermined significance (ASC-US) constituted the study specimens. Of the 168 ASC-US samples, 134 (79.8%) were from Saudi patients. The hc2 test was positive in 33 (19.6%) of the total patients, 20% among Saudi patients, and 17.6% among non-Saudi patients. Xpert(®) HPV produced positive results in 30 (17.8%) of the samples. The overall concordance rate between the two tests was 98.2%, and the positive concordance rate was 91%. There were three samples tested positive by hc2 that tested negative by Xpert(®) HPV. HPV 16, HPV 18/45 and HPV other types were the most common types. Both tests have a reasonable positive concordance rate, although hc2 detected more cases than Xpert(®) HPV. Xpert(®) HPV provides a viable alternative to the hc2 test with similar detection results for samples with ASC-US.

  19. Ricerca di HPV-DNA e tipizzazione virale nella diagnostica di prevenzione del carcinoma della cervice uterina

    Directory of Open Access Journals (Sweden)

    Mauro Carcheri

    2003-03-01

    Full Text Available Papillomaviruses are relatively ubiquitous and have been described as causative agents for epithelial lesions in a wide variety of animals as well as in humans. Approximately 30 HPV types have been isolated from anogenital epithelium (cervix, vagina, vulva, rectum and penis. HPVs induce a variety of proliferative lesions, but only the “high-risk” types are associated with anogenital cancers. “Low-risk” viral types include HPV-6 and HPV-11; “high-risk” types include HPV-16, 18, 31 and 56. HPVs “high-risk” types are more strongly associated with high-grade lesions (HSIL than they are with low-grade lesions (LSIL. The traditional process for cervical cancer screening programs (PAP test is vulnerable to air drying artifacts and has limits to sensitivity, since as many as 90 percent of collectet cells can be discarted with collection device. The present study was undertaken to assess the screening performance of HPV-DNA typing in a sample of 142 women drawn from a routine screening for the prevention of cervical cancer. The results indicate that HPV-DNA screening and typing, used together with PAP test, can improve the detection of patients with cervical disease and can serve as a quality assurance indicator in cervical cancer screening programs.

  20. Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays.

    Science.gov (United States)

    Iftner, Thomas; Germ, Liesje; Swoyer, Ryan; Kjaer, Susanne Kruger; Breugelmans, J Gabrielle; Munk, Christian; Stubenrauch, Frank; Antonello, Joseph; Bryan, Janine T; Taddeo, Frank J

    2009-07-01

    Human papillomavirus (HPV) DNA genotyping is an essential test to establish efficacy in HPV vaccine clinical trials and HPV prevalence in natural history studies. A number of HPV DNA genotyping methods have been cited in the literature, but the comparability of the outcomes from the different methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example, were 0.806, 0.646, 0.920, and 0.860, respectively; the specificities were 0.986, 0.998, 0.960, and 0.986, respectively. The overall comparability of detection of the 15 HPV types was quite high. Analyses of DNA genotype testing compared to cytology results demonstrated a significant discordance between cytology-negative (normal) and HPV DNA-positive results. This demonstrates the challenges of cytological diagnosis and the possibility that a significant number of HPV-infected cells may appear cytologically normal.

  1. The Amerindian mtDNA haplogroup B2 enhances the risk of HPV for cervical cancer: de-regulation of mitochondrial genes may be involved.

    Science.gov (United States)

    Guardado-Estrada, Mariano; Medina-Martínez, Ingrid; Juárez-Torres, Eligia; Roman-Bassaure, Edgar; Macías, Luis; Alfaro, Ana; Alcántara-Vázquez, Avissai; Alonso, Patricia; Gomez, Guillermo; Cruz-Talonia, Fernando; Serna, Luis; Muñoz-Cortez, Sergio; Borges-Ibañez, Manuel; Espinosa, Ana; Kofman, Susana; Berumen, Jaime

    2012-04-01

    Although human papillomavirus (HPV) infection is the main causal factor for cervical cancer (CC), there are data suggesting that genetic factors could modulate the risk for CC. Sibling studies suggest that maternally inherited factors could be involved in CC. To assess whether mitochondrial DNA (mtDNA) polymorphisms are associated to CC, HPV infection and HPV types, a case-control study was performed in the Mexican population. Polymorphism of mtDNA D-loop was investigated in 187 CC patients and 270 healthy controls. HPV was detected and typed in cervical scrapes. The expression of 29 mitochondrial genes was analyzed in a subset of 45 tumor biopsies using the expression microarray ST1.0. The Amerindian haplogroup B2 increased the risk for CC (odds ratio (OR)=1.6; 95% confidence interval (CI): 1.05-2.58) and enhanced 36% (OR=208; 95% CI: 25.2-1735.5) the risk conferred by the HPV alone (OR=152.9; 95% CI: 65.4-357.5). In cases, the distribution of HPV types was similar in all haplogroups but one (D1), in which is remarkable the absence of HPV18, a very low frequency of HPV16 and high frequencies of HPV45, HPV31 and other HPV types. Two mtDNA genes (mitochondrial aspartic acid tRNA (MT-TD), mitochondrial lysine tRNA (MT-TK)) could be involved in the increased risk conferred by the haplogroup B2, as they were upregulated exclusively in B2 tumors (P<0.01, t-test). Although the association of mtDNA with CC and HPV infection is clear, other studies with higher sample size will be needed to elucidate the role of mtDNA in cervical carcinogenesis.

  2. Triage of HR-HPV positive women with minor cytological abnormalities: a comparison of mRNA testing, HPV DNA testing, and repeat cytology using a 4-year follow-up of a population-based study.

    Directory of Open Access Journals (Sweden)

    Maria Persson

    Full Text Available OBJECTIVE: Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. METHODS: Liquid-based (PreservCyt cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS and low-grade squamous intraepithelial lesions (LSIL within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA (Gene-Probe Inc., San Diego, CA, USA. Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center. RESULTS: Nine of 25 (36% women in the ASCUS group, and 64 of 180 (36% women in the LSIL group developed cervical intraepithelial neoplasia (CIN grade 2 or worse during 4 years of follow-up. 162 (74% women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100% and LSIL (78.1 and 75.8% groups, although specificity was insufficient (<50%. HPV16 DNA testing and repeat cytology were more specific than APTIMA. CONCLUSION: The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL.

  3. 高危型HPV-DNA检测对ASCUS分流监测的临床价值%Value of High-risk HPV DNA Testing in the Triage of ASCUS

    Institute of Scientific and Technical Information of China (English)

    朱熠; 张国楠; 姚宇琪

    2012-01-01

    Objective; To evaluate high-risk human papilomavims DNA (HR-HPV DNA) testing in triage of women with a-typical squamous cells of undetermined significance (ASCUS). Methods; A total of 376 women with a diagnosis of ASCUS from the primary screening were selected for triage. Their cervical smears were collected and evaluated by using conventional cytological examination in combination with a high-risk HPV-DNA test ( hybrid capture 2) . The women were categorized into four groups; Group A, Cytology + /HPV + ; Group B, Cytology - /HPV + ; Group C , Cytology + /HPV - ; and Group D, Cytology - /HPV - . Women within Group A-C were admitted for colposcopy and cervical biopsy. Group D was considered as a low-risk group for tumor development, and was not brought into comparison. Results; In women in Group A(n = 113) , the prevalence of histological verified CIN Ⅱ/Ⅲ was 41.59% , in Group B (n = 80) 23.75% , and in Group C (n = 27) 3.70%. The prevalence of a high-risk HPV infection decreased with age in women with ASCUS. It was 71. 43% in women < 30 years and 26. 80% in women ≥50 years. Conclusion; Adding a high-risk HPV-DNA test in secondary screening increases the identification of women with CIN Ⅱ/Ⅲ lesions by 46. 15% in comparison with repeat cytology. The clinical significance of the ASCUS diagnosis varies with age of the women. HR-HPV DNA testing is a feasible method for triage of ASCUS.%目的 评价高危型人类乳头瘤病毒(HPV)检测对未明确意义的不典型鳞状细胞(Atypical squamous cells of undetermined significance,ASCUS) 分流监测的临床价值.方法 对376例初筛结果为ASCUS病例,3个月后进行宫颈液基细胞学复查和高危型HPV-DNA检测,并根据检查结果将病例分为4组:A组,细胞学+/HPV+;B组,细胞学-/HPV+;C组,细胞学+/HPV-;D组,细胞学-/HPV-.对A、B、C组病例进行了阴道镜检查及活检,比较3组间宫颈上皮内瘤变(cervical intraepithelial neoplasma,CIN)发生率的差异.由于D组发

  4. Automated extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas

    2011-01-01

    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing t...

  5. Antitumor Response to a Codon-Optimized HPV-16 E7/HSP70 Fusion Antigen DNA Vaccine.

    Science.gov (United States)

    Soleimanjahi, Hoorieh; Razavinikoo, Hadi; Fotouhi, Fatemeh; Ardebili, Abdollah

    2017-09-01

    Vaccines based on virus-like particles are effective against Human Papilloma Virus (HPV) infection; however, they have not shown a therapeutic effect against HPV-associated diseases. New immunotherapy strategies based on immune responses against tumor antigens can positively affect the clearance of HPV-associated lesions. To generate two therapeutic fusion DNA vaccines (optimizedE7/mouseHSP70 and wildE7/mouseHSP70) to induce antitumor specific responses in mice models. Mice were immunized with recombinant DNA vaccines. The splenocytes of immunized mice were collected and lactate dehydrogenase and IFN-γ productions were measured after three injections in order to evaluate cytotoxic T lymphocytes (CTLs) activity. MTT assay was carried out for lymphocyte stimulation. The fusion DNA vaccines, specifically uE7-HSP70, elicited varying levels of IFN-γ and CTLs responses compared to the control group (P<0.05). Furthermore, antitumor response and tumor size reduction in fusion DNA vaccines groups were significantly higher than in the negative control group (P<0.05). It is concluded that our fusion DNA vaccines considerably enhanced specific cellular responses against HPV tumor model. In addition, optimized E7 showed a notable immunogenicity and inhibitory effect on the reduction of tumor size.

  6. Photon-induced cell migration and integrin expression promoted by DNA integration of HPV16 genome

    Energy Technology Data Exchange (ETDEWEB)

    Rieken, Stefan; Simon, Florian; Habermehl, Daniel; Dittmar, Jan Oliver; Combs, Stephanie E.; Weber, Klaus; Debus, Juergen; Lindel, Katja [University Hospital of Heidelberg, Department of Radiation Therapy and Radiation Oncology, Heidelberg (Germany)

    2014-10-15

    Persistent human papilloma virus 16 (HPV16) infections are a major cause of cervical cancer. The integration of the viral DNA into the host genome causes E2 gene disruption which prevents apoptosis and increases host cell motility. In cervical cancer patients, survival is limited by local infiltration and systemic dissemination. Surgical control rates are poor in cases of parametrial infiltration. In these patients, radiotherapy (RT) is administered to enhance local control. However, photon irradiation itself has been reported to increase cell motility. In cases of E2-disrupted cervical cancers, this phenomenon would impose an additional risk of enhanced tumor cell motility. Here, we analyze mechanisms underlying photon-increased migration in keratinocytes with differential E2 gene status. Isogenic W12 (intact E2 gene status) and S12 (disrupted E2 gene status) keratinocytes were analyzed in fibronectin-based and serum-stimulated migration experiments following single photon doses of 0, 2, and 10 Gy. Quantitative FACS analyses of integrin expression were performed. Migration and adhesion are increased in E2 gene-disrupted keratinocytes. E2 gene disruption promotes attractability by serum components, therefore, effectuating the risk of local infiltration and systemic dissemination. In S12 cells, migration is further increased by photon RT which leads to enhanced expression of fibronectin receptor integrins. HPV16-associated E2 gene disruption is a main predictor of treatment-refractory cancer virulence. E2 gene disruption promotes cell motility. Following photon RT, E2-disrupted tumors bear the risk of integrin-related infiltration and dissemination. (orig.) [German] Persistierende Infektionen mit humanen Papillomaviren 16 (HPV16) sind ein Hauptausloeser des Zervixkarzinoms. Die Integration der viralen DNS in das Wirtszellgenom fuehrt zum Integritaetsverlust des E2-Gens, wodurch in der Wirtszelle Apoptose verhindert und Motilitaet gesteigert werden. In

  7. HPV-FASTER

    DEFF Research Database (Denmark)

    Bosch, F Xavier; Robles, Claudia; Díaz, Mireia;

    2016-01-01

    Human papillomavirus (HPV)-related screening technologies and HPV vaccination offer enormous potential for cancer prevention, notably prevention of cervical cancer. The effectiveness of these approaches is, however, suboptimal owing to limited implementation of screening programmes and restricted...... indications for HPV vaccination. Trials of HPV vaccination in women aged up to 55 years have shown almost 90% protection from cervical precancer caused by HPV16/18 among HPV16/18-DNA-negative women. We propose extending routine vaccination programmes to women of up to 30 years of age (and to the 45-50-year...... age groups in some settings), paired with at least one HPV-screening test at age 30 years or older. Expanding the indications for HPV vaccination and much greater use of HPV testing in screening programmes has the potential to accelerate the decline in cervical cancer incidence. Such a combined...

  8. Double positivity for HPV DNA/p16 in tonsillar and base of tongue cancer improves prognostication

    DEFF Research Database (Denmark)

    Garnaes, Emilie; Frederiksen, Kirsten; Kiss, Katalin

    2016-01-01

    of tongue squamous cell carcinoma (BSCC) when stratifying for HPV DNA status, p16 expression and combined HPV/p16 status. We included all patients (n = 797) diagnosed with TSCCs and BSCCs in Eastern Denmark as registered in the Danish Head and Neck Cancer Group (DAHANCA) database and the Danish Pathology...... Databank, 2000–2010. Patients were treated according to national guidelines (radiotherapy +/− concomitant cisplatin). All specimens were analysed using HPV DNA PCR and p16 immunohistochemistry. Clinical information was retrieved from the DAHANCA database and the Danish National Patient Registry....... Information on vital status was obtained from the Danish Civil Registration System. We observed improved OS for HPV+/p16+ BSCCs compared to HPV−/p16− (hazard ratio for death [HR], 0.15; 95% CI, 0.09–0.24). Among STSCCs, HPV+/p16+ showed the lowest HR (0.19, 95% CI, 0.13–0.29); whereas, HPV−/p16+ showed...

  9. The Relationship between Syntactic Structure Analysis Features, Histological Grade and High-Risk HPV DNA in Cervical Intraepithelial Neoplasia

    Directory of Open Access Journals (Sweden)

    Arnold‐Jan Kruse

    2004-01-01

    Full Text Available Aim: To assess the correlation between syntactic structure analysis (SSA features, revised dysplasia grade and the presence of high‐risk human papillomavirus DNA in cervical intraepithelial neoplasia (CIN. Materials and methods: HPV polymerase chain reaction (PCR was assessed in 101 consecutive biopsies and consensus in CIN grade between the experts occurred in 88 cases (CIN1=16, CIN2=27, CIN3=45. SSA was performed in the diagnostic histological section of the CIN lesions in these patients and SSA features were compared with the blind review CIN grade, and presence/absence of high‐risk HPV DNA. Results: One of the SSA features (points from which the surrounding surfaces has 4 edges, PECO‐4 was significantly different between all three consensus CIN grades. Many more features revealed significant differences between CIN1 and CIN2 or between CIN2 and CIN3 cases. With stepwise discriminant analysis, the best multivariate combination of features to distinguish the different CIN grades were the Maximum MST Line Length (MML and the Area Disorder. Crude overall classification of the consensus grades with these features was 69%. The MML and the Area Disorder is also the best combination to distinguish cases with and without high‐risk HPV DNA (77% correct classifications. Conclusions: SSA features are correlated with both CIN grade and presence of high‐risk HPV DNA, but the discrimination power is not good enough to be used as a routine method for quality control of subjective grade or as a surrogate marker for high‐risk HPV DNA presence.

  10. HPV DNA testing in population-based cervical screening (VUSA-Screen study) : results and implications

    NARCIS (Netherlands)

    Rijkaart, D. C.; Berkhof, J.; van Kemenade, F. J.; Coupe, V. M. H.; Rozendaal, L.; Heideman, D. A. M.; Verheijen, R. H. M.; Bulk, S.; Verweij, W.; Snijders, P. J. F.; Meijer, C. J. L. M.

    2012-01-01

    BACKGROUND: Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrHPV) testing in routine screening. METHODS: In all, 25 871 women (29-61) enrolled in our population-based

  11. HPV DNA testing in population-based cervical screening (VUSA-Screen study) : results and implications

    NARCIS (Netherlands)

    Rijkaart, D. C.; Berkhof, J.; van Kemenade, F. J.; Coupe, V. M. H.; Rozendaal, L.; Heideman, D. A. M.; Verheijen, R. H. M.; Bulk, S.; Verweij, W.; Snijders, P. J. F.; Meijer, C. J. L. M.

    2012-01-01

    BACKGROUND: Human papillomavirus (HPV) testing is more sensitive than cytology for detecting high-grade cervical intraepithelial neoplasia (CIN). We evaluated the performance of high-risk HPV (hrHPV) testing in routine screening. METHODS: In all, 25 871 women (29-61) enrolled in our population-based

  12. Double positivity for HPV DNA/p16 in tonsillar and base of tongue cancer improves prognostication: Insights from a large population-based study.

    Science.gov (United States)

    Garnaes, Emilie; Frederiksen, Kirsten; Kiss, Katalin; Andersen, Luise; Therkildsen, Marianne H; Franzmann, Maria B; Specht, Lena; Andersen, Elo; Norrild, Bodil; Kjaer, Susanne K; von Buchwald, Christian

    2016-12-01

    The aim was to explore the overall survival (OS) for palatine tonsillar squamous cell carcinoma (TSCC), subdivided, according to certainty of tonsillar tumour origin, into specified tonsillar squamous cell carcinomas (STSCCs) and nonspecified tonsillar squamous cell carcinomas (NSTSCCs), and base of tongue squamous cell carcinoma (BSCC) when stratifying for HPV DNA status, p16 expression and combined HPV/p16 status. We included all patients (n = 797) diagnosed with TSCCs and BSCCs in Eastern Denmark as registered in the Danish Head and Neck Cancer Group (DAHANCA) database and the Danish Pathology Databank, 2000-2010. Patients were treated according to national guidelines (radiotherapy +/- concomitant cisplatin). All specimens were analysed using HPV DNA PCR and p16 immunohistochemistry. Clinical information was retrieved from the DAHANCA database and the Danish National Patient Registry. Information on vital status was obtained from the Danish Civil Registration System. We observed improved OS for HPV+/p16+ BSCCs compared to HPV-/p16- (hazard ratio for death [HR], 0.15; 95% CI, 0.09-0.24). Among STSCCs, HPV+/p16+ showed the lowest HR (0.19, 95% CI, 0.13-0.29); whereas, HPV-/p16+ showed an intermediate HR (0.39; 95% CI, 0.22-0.70). For NSTSCCs, HPV+/p16+ and HPV-/p16+ showed similar OS (HRs, 0.39; 95% CI, 0.26-0.59; and 0.48; 95% CI, 0.24-0.95, respectively). Combined HPV+/p16+ was a significantly better prognostic marker in BSCCs and STSCCs than HPV DNA and p16, alone (all p-values p16 (p = 0.53), alone. In conclusion, double positivity for HPV/p16 in conjunction with the certainty of tumour site improved prognosis.

  13. Physical state and expression of HPV DNA in benign and dysplastic cervical tissue: different levels of viral integration are correlated with lesion grade.

    Science.gov (United States)

    Hudelist, Gernot; Manavi, Mahmood; Pischinger, Kerstin I D; Watkins-Riedel, Thomas; Singer, Christian F; Kubista, E; Czerwenka, Klaus F

    2004-03-01

    Human papillomavirus (HPV) infection is the most important event in the malignant transformation of human cervical epithelium. Several high-risk (HR-)HPV subtypes have been identified, which lead to CIN and subsequently to invasive carcinoma. The reason for this phenomenon is still unknown, but it seems to be related to the physical state of HPV DNA. Digene HC II test was used to identify HR- and/or low-risk (LR-)HPV infections in cervical swabs of 275 women attending our clinic for routine cytological screening and/or colposcopy because of an abnormal Pap smear comprising low-grade squamous intraepithelial lesions (LGSIL) and high-grade SIL (HGSIL). Specific HR (16, 18, 31, 33, 52b, 58) and LR (6, 11) subtypes were characterized in cervical biopsies of 10 women with benign cellular changes and of 68 women with CIN I-III by the PCR-restriction enzyme method. The physical state of HPV DNA (episomal, mixed and integrated form) was analyzed by bi-dimensional (2D)-gel electrophoresis. In addition, mRNA expression of E6/E7 genes was analyzed by RT-PCR. Furthermore, the relative virus load was determined in nine selected cases. The physical state and transcriptional activity of HPV DNA were then correlated to histopathological results. LR-HPV infection [27 cases (9.8%)] and HR-HPV infection [121 cases (44%)] of cervical swabs were clearly correlated to the degree of SIL. Further HPV typing in cervical biopsies of 78 women showed that HPV6 and 11 were restricted to benign cellular changes, CIN I and II, whereas HPV16 and 18 were observed predominantly in CIN III/CIS (P=0.01). No clear distribution pattern was observed for HPV31, 33, 52b and 58. Expression of HPV E6 and E7 transcripts was uniformly correlated with the different physical state of HPV DNA. Analyzing the physical state of these HPV subtypes, HPV6 and 11 could only be detected as an episomal form, independent of SIL grade. In normal epithelium and in CIN I and II, HPV16 and 18 were exclusively found in the

  14. Triage of HR-HPV Positive Women with Minor Cytological Abnormalities: A Comparison of mRNA Testing, HPV DNA Testing, and Repeat Cytology Using a 4-Year Follow-Up of a Population-Based Study

    Science.gov (United States)

    Persson, Maria; Elfström, K. Miriam; Brismar Wendel, Sophia; Weiderpass, Elisabete; Andersson, Sonia

    2014-01-01

    Objective Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV) is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA) to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. Methods Liquid-based (PreservCyt) cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA (Gene-Probe Inc., San Diego, CA, USA). Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center. Results Nine of 25 (36%) women in the ASCUS group, and 64 of 180 (36%) women in the LSIL group developed cervical intraepithelial neoplasia (CIN) grade 2 or worse during 4 years of follow-up. 162 (74%) women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100%) and LSIL (78.1 and 75.8%) groups, although specificity was insufficient (cytology were more specific than APTIMA. Conclusion The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL. PMID:24587193

  15. Triage of HR-HPV positive women with minor cytological abnormalities: a comparison of mRNA testing, HPV DNA testing, and repeat cytology using a 4-year follow-up of a population-based study.

    Science.gov (United States)

    Persson, Maria; Elfström, K Miriam; Brismar Wendel, Sophia; Weiderpass, Elisabete; Andersson, Sonia

    2014-01-01

    Expression of the viral E6/E7 oncogenes of high-risk human papillomaviruses (HR-HPV) is necessary for malignant conversion and maintenance in cervical tissue. In order to determine whether HR-HPV E6/E7 mRNA testing more effectively predicts precancerous lesions and invasive cervical cancer than HR-HPV DNA testing, we aimed to compare triage using HR-HPV E6/E7 mRNA testing by APTIMA HPV Assay (APTIMA) to HPV16 DNA testing, HPV16/18 DNA testing, and repeat cytology. Liquid-based (PreservCyt) cell samples were obtained from HR-HPV-positive women diagnosed with atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesions (LSIL) within the framework of the population-based cervical cancer screening program in Stockholm, Sweden. Samples were tested for HR-HPV E6/E7 mRNA by APTIMA (Gene-Probe Inc., San Diego, CA, USA). Women were followed up for 4 years after the index cytology via medical and laboratory records, and the Stockholm Oncology Center. Nine of 25 (36%) women in the ASCUS group, and 64 of 180 (36%) women in the LSIL group developed cervical intraepithelial neoplasia (CIN) grade 2 or worse during 4 years of follow-up. 162 (74%) women were APTIMA-positive, and APTIMA had the highest sensitivity to predict CIN2 or worse and CIN3 or worse in the ASCUS (77.8% and 100%) and LSIL (78.1 and 75.8%) groups, although specificity was insufficient (cytology were more specific than APTIMA. The results of this population-based study with comprehensive follow-up support the use of APTIMA as a triage test for women with ASCUS. More focused investigation is required for women with LSIL.

  16. Nondestructive DNA extraction from museum specimens.

    Science.gov (United States)

    Hofreiter, Michael

    2012-01-01

    Natural history museums around the world hold millions of animal and plant specimens that are potentially amenable to genetic analyses. With more and more populations and species becoming extinct, the importance of these specimens for phylogenetic and phylogeographic analyses is rapidly increasing. However, as most DNA extraction methods damage the specimens, nondestructive extraction methods are useful to balance the demands of molecular biologists, morphologists, and museum curators. Here, I describe a method for nondestructive DNA extraction from bony specimens (i.e., bones and teeth). In this method, the specimens are soaked in extraction buffer, and DNA is then purified from the soaking solution using adsorption to silica. The method reliably yields mitochondrial and often also nuclear DNA. The method has been adapted to DNA extraction from other types of specimens such as arthropods.

  17. Effect of glacial acetic acid treatment of cervical ThinPrep specimens on HPV DNA detection with the cobas 4800 HPV test.

    Science.gov (United States)

    McMenamin, M; McKenna, M

    2013-10-01

    Cytology laboratories in the UK routinely treat unsatisfactory cervical liquid-based cytology (LBC) specimens with glacial acetic acid (GAA) to reduce the unsatisfactory rate. However, there is limited published data on the effect of GAA reprocessing on the molecular detection of human papillomavirus (HPV). The aim of this study was to assess the impact of GAA treatment of cervical ThinPrep(®) samples on HPV detection with the cobas(®) 4800 HPV Test (Roche Molecular Systems, Pleasanton, CA, USA). Residual ThinPrep samples (n = 121) were selected to provide a range of typical cytology results and enrich the study samples for HPV positivity. Specimens were equally split into two fractions: one part treated with 10% GAA and the other part left untreated. All samples were HPV tested using the cobas 4800 HPV Test, which simultaneously detects a total of 14 high-risk HPV (hrHPV) genotypes and individually identifies HPV16 and HPV18. The HPV positive/negative status of tested samples determined the level of agreement between treated and untreated fractions; one sample failed owing to detection of a clot by the instrument during pipetting, leaving 120 samples in the study. Statistical analysis was performed using an unweighted kappa. Analysis of overall HPV positivity showed 97.5% (117/120) agreement between the treated and untreated fractions with a kappa value of 0.95. There were 63/65 (96.9%) concordant HPV positive and 54/55 (98.2%) concordant HPV negative results. In addition to the three discordant results for overall HPV positivity, there were three HPV type-specific discrepancies giving a total of 114/120 concordant HPV results (95% agreement). Glacial acetic acid (GAA) treatment of cervical ThinPrep specimens does not have significant adverse affects on HPV detection with the cobas 4800 HPV Test. © 2013 John Wiley & Sons Ltd.

  18. HPV 5 and 8 E6 abrogate ATR activity resulting in increased persistence of UVB induced DNA damage.

    Directory of Open Access Journals (Sweden)

    Nicholas A Wallace

    Full Text Available The role of the E6 oncoprotein from high-risk members of the α human papillomavirus genus in anogenital cancer has been well established. However, far less is known about the E6 protein from the β human papillomavirus genus (β-HPVs. Some β-HPVs potentially play a role in non-melanoma skin cancer development, although they are not required for tumor maintenance. Instead, they may act as a co-factor that enhances the carcinogenic potential of UV damage. Indeed, the E6 protein from certain β-HPVs (HPV 5 and 8 promotes the degradation of p300, a histone acetyl transferase involved in UV damage repair. Here, we show that the expression of HPV 5 and 8 E6 increases thymine dimer persistence as well as the likelihood of a UVB induced double strand break (DSB. Importantly, we provide a mechanism for the increased DNA damage by showing that both extended thymine dimer persistence as well as elevated DSB levels are dependent on the ability of HPV 8 E6 to promote p300 degradation. We further demonstrate that HPV 5 and 8 E6 expression reduces the mRNA and protein levels of ATR, a PI3 kinase family member that plays a key role in UV damage signaling, but that these levels remain unperturbed in cells expressing a mutated HPV 8 E6 incapable of promoting p300 degradation. We confirm that the degradation of p300 leads to a reduction in ATR protein levels, by showing that ATR levels rebound when a p300 mutant resistant to HPV 8 mediated degradation and HPV 8 E6 are co-transfected. Conversely, we show that ATR protein levels are reduced when p300 is targeted for degradation by siRNA. Moreover, we show the reduced ATR levels in HPV 5 and 8 E6 expressing cells results in delayed ATR activation and an attenuated ability of cells to phosphorylate, and as a result accumulate, p53 in response to UVB exposure, leading to significantly reduced cell cycle arrest. In conclusion, these data demonstrate that β-HPV E6 expression can enhance the carcinogenic potential of

  19. Post hoc analysis of the PATRICIA randomized trial of the efficacy of human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against incident and persistent infection with nonvaccine oncogenic HPV types using an alternative multiplex type-specific PCR assay for HPV DNA.

    Science.gov (United States)

    Struyf, Frank; Colau, Brigitte; Wheeler, Cosette M; Naud, Paulo; Garland, Suzanne; Quint, Wim; Chow, Song-Nan; Salmerón, Jorge; Lehtinen, Matti; Del Rosario-Raymundo, M Rowena; Paavonen, Jorma; Teixeira, Júlio C; Germar, Maria Julieta; Peters, Klaus; Skinner, S Rachel; Limson, Genara; Castellsagué, Xavier; Poppe, Willy A J; Ramjattan, Brian; Klein, Terry D; Schwarz, Tino F; Chatterjee, Archana; Tjalma, Wiebren A A; Diaz-Mitoma, Francisco; Lewis, David J M; Harper, Diane M; Molijn, Anco; van Doorn, Leen-Jan; David, Marie-Pierre; Dubin, Gary

    2015-02-01

    The efficacy of the human papillomavirus type 16 (HPV-16)/HPV-18 AS04-adjuvanted vaccine against cervical infections with HPV in the Papilloma Trial against Cancer in Young Adults (PATRICIA) was evaluated using a combination of the broad-spectrum L1-based SPF10 PCR-DNA enzyme immunoassay (DEIA)/line probe assay (LiPA25) system with type-specific PCRs for HPV-16 and -18. Broad-spectrum PCR assays may underestimate the presence of HPV genotypes present at relatively low concentrations in multiple infections, due to competition between genotypes. Therefore, samples were retrospectively reanalyzed using a testing algorithm incorporating the SPF10 PCR-DEIA/LiPA25 plus a novel E6-based multiplex type-specific PCR and reverse hybridization assay (MPTS12 RHA), which permits detection of a panel of nine oncogenic HPV genotypes (types 16, 18, 31, 33, 35, 45, 52, 58, and 59). For the vaccine against HPV types 16 and 18, there was no major impact on estimates of vaccine efficacy (VE) for incident or 6-month or 12-month persistent infections when the MPTS12 RHA was included in the testing algorithm versus estimates with the protocol-specified algorithm. However, the alternative testing algorithm showed greater sensitivity than the protocol-specified algorithm for detection of some nonvaccine oncogenic HPV types. More cases were gained in the control group than in the vaccine group, leading to higher point estimates of VE for 6-month and 12-month persistent infections for the nonvaccine oncogenic types included in the MPTS12 RHA assay (types 31, 33, 35, 45, 52, 58, and 59). This post hoc analysis indicates that the per-protocol testing algorithm used in PATRICIA underestimated the VE against some nonvaccine oncogenic HPV types and that the choice of the HPV DNA testing methodology is important for the evaluation of VE in clinical trials. (This study has been registered at ClinicalTrials.gov under registration no. NCT00122681.). Copyright © 2015, American Society for

  20. Human papillomavirus (HPV) genotyping of cutaneous warts in Greek children.

    Science.gov (United States)

    Giannaki, Maria; Kakourou, Talia; Theodoridou, Maria; Syriopoulou, Vassiliki; Kabouris, Marios; Louizou, Eirini; Chrousos, George

    2013-01-01

    The human papillomavirus (HPV) infects the squamous epithelium of the skin and produces common warts, plantar warts, and flat warts, which occur commonly on the hands, face, and feet. The objective of this study was to determine the presence of HPV in warts in children in order to associate the virus with the disease. Sixty-eight children with clinically diagnosed cutaneous warts were recruited. Skin biopsy samples were examined and DNA was extracted using a commercially available kit. To distinguish between the HPV types, we used a specific pair of primers to amplify the HPV DNA. Polymerase chain reaction amplification of the L1 region was followed by restriction fragment length polymorphism analysis and Luminex xMAP technology. HPV 57 was the predominant type in our study, although the detection of the high-risk HPV type 16 in 33% of our positive samples indicates the presence of mucosal high-risk HPV types in the skin of children. It seems that the newly introduced Luminex assay maximized the discrimination of genotypes even in the case of multiple HPV infections. Or findings also suggest the presence of high-risk HPV types in cutaneous warts. © 2013 Wiley Periodicals, Inc.

  1. Extracting DNA from submerged pine wood.

    Science.gov (United States)

    Reynolds, M Megan; Williams, Claire G

    2004-10-01

    A DNA extraction protocol for submerged pine logs was developed with the following properties: (i) high molecular weight DNA, (ii) PCR amplification of chloroplast and nuclear sequences, and (iii) high sequence homology to voucher pine specimens. The DNA extraction protocol was modified from a cetyltrimehtylammonium bromide (CTAB) protocol by adding stringent electrophoretic purification, proteinase K, RNAse, polyvinyl pyrrolidone (PVP), and Gene Releaser. Chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase) could be amplified. Nuclear ribosomal sequences had >95% homology to Pinus taeda and Pinus palustris. Microsatellite polymorphism for PtTX2082 matched 2 of 14 known P. taeda alleles. Our results show DNA analysis for submerged conifer wood is feasible.

  2. A DNA vaccine encoding mutated HPV58 mE6E7-Fc-GPI fusion antigen and GM-CSF and B7.1

    Directory of Open Access Journals (Sweden)

    Wang H

    2015-10-01

    Full Text Available He Wang,1 Jiyun Yu,2 Li Li1 1Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi, 2Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing, People’s Republic of China Background: Persistent infection with high-risk human papillomavirus (HPV is a predominant cause of cervical cancer, and HPV58 is the third most common virus detected in the patients with cervical cancer in Asia. E6 and E7 are the viral oncogenes which are constitutively expressed in HPV-associated tumor cells and can be used as target antigens for related immunotherapy. In this study, we modified the HPV58 E6 and E7 oncogenes to eliminate their oncogenic potential and constructed a recombinant DNA vaccine that coexpresses the sig-HPV58 mE6E7-Fc-GPI fusion antigen in addition to granulocyte-macrophage colony-stimulating factor (GM-CSF and B7.1 as molecular adjuvants (PVAX1-HPV58 mE6E7FcGB for the treatment of HPV58 (+ cancer. Methods: PVAX1-HPV58 mE6E7FcGB recombinant DNA vaccine was constructed to express a fusion protein containing a signal peptide, a modified HPV58 mE6E7 gene, and human IgG Fc and glycosylphosphatidylinositol (GPI-anchoring sequences using the modified DNA vaccine vector PVAX1-IRES-GM/B7.1 that coexpresses GM-CSF, and B7.1. C57BL/6 mice were challenged by HPV58 E6E7-expressing B16-HPV58 E6E7 cells, followed by immunization by PVAX1-HPV58 mE6E7FcGB vaccine on days 7, 14, 21 after tumor challenge. The cellular immune responses in immunized mice were assessed by measuring IFN-γ production in splenocytes upon stimulation by HPV58 E6E7-GST protein and the lysis of B16-HPV58 E6E7 target cells by splenocytes after restimulation with HPV58 E6E7-GST protein. The antitumor efficacy was evaluated by monitoring the growth of the tumor. Results: PVAX1-HPV58 mE6E7FcGB elicited varying levels of IFN-lsgdB58onn T-cell immune responses and lysis of target cell in mice in response to the

  3. High-Risk Human Papillomavirus (hrHPV) E6/E7 mRNA Testing by PreTect HPV-Proofer for Detection of Cervical High-Grade Intraepithelial Neoplasia and Cancer among hrHPV DNA-Positive Women with Normal Cytology

    Science.gov (United States)

    Rijkaart, D. C.; Heideman, D. A. M.; Coupe, V. M. H.; Brink, A. A. T. P.; Verheijen, R. H. M.; Skomedal, H.; Karlsen, F.; Morland, E.; Snijders, P. J. F.

    2012-01-01

    Our aim was to investigate whether high-risk HPV (hrHPV) mRNA detection by PreTect HPV-Proofer can be used to stratify hrHPV DNA-positive women of different cytology classes for risk of high-grade cervical intraepithelial neoplasia or worse (cervical precancer or cancer, i.e., cervical intraepithelial neoplasia grade 2 or higher [≥CIN2]). A total of 375 women participating in population-based screening, with a GP5+/6+-PCR hrHPV DNA-positive cervical scrape with normal cytology (n = 202), borderline or mild dyskaryosis (BMD) (n = 88), or moderate dyskaryosis or worse (>BMD) (n = 85), were enrolled. Cervical scrapes were additionally subjected to HPV16/18/31/33/45 E6/E7 mRNA analysis by PreTect HPV-Proofer (mRNA test). Referral and follow-up policies were based on cytology, hrHPV DNA, and mRNA testing. The primary study endpoint was the number of ≥CIN2 detected within 3 years of follow-up. The mRNA positivity increased with the severity of cytological abnormality, ranging from 32% (64/202) in hrHPV DNA-positive women with normal cytology to 47% (41/88) in BMD and 68% (58/85) in >BMD groups (P cytology, i.e., 0.55 (95% confidence interval [95% CI], 0.34 to 0.76) in mRNA-positive versus 0.20 (95% CI, 0.07 to 0.33) in mRNA-negative women. In hrHPV DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the ≥CIN2 risk. In conclusion, mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV DNA-positive women with normal cytology in need of immediate referral for colposcopy. PMID:22553244

  4. Human papillomavirus DNA in plasma of patients with HPV16 DNA-positive uterine cervical cancer.

    OpenAIRE

    Shimada, Takako; Yamaguchi, Naohiro; Nishida, Noriyuki; Yamasaki, Kentaro; Miura, Kiyonori; Katamine, Shigeru; Masuzaki, Hideaki

    2010-01-01

    OBJECTIVES: The squamous cell carcinoma antigen is considered the most accurate serologic tumor marker for uterine cervical carcinoma. However, serum squamous cell carcinoma antigen levels were found to correlate significantly with clinical severity of atopic dermatitis and chronic renal failure. The present study was conducted in patients with human papillomavirus 16 DNA-positive uterine cervical cancer to determine the plasma level of human papillomavirus 16 DNA and the diagnostic values of...

  5. DNA extraction from keratin and chitin.

    Science.gov (United States)

    Campos, Paula F; Gilbert, Thomas M P

    2012-01-01

    DNA extracted from keratinous and chitinous materials can be a useful source of genetic information. To effectively liberate the DNA from these materials, buffers containing relatively high levels of DTT, proteinase K, and detergent are recommended, followed by purification using either silica-column or organic methods.

  6. [Application of the QIAamp DNA Investigator Kit and Prepfiler Forensic DNA Extraction Kit in genomic DNA extraction from skeletal remains].

    Science.gov (United States)

    Ludwikowska-Pawłowska, Małgorzata; Jacewicz, Renata; Jedrzejczyk, Maciej; Prośniak, Adam; Berent, Jarosław

    2009-01-01

    The report presents an application of the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit in genomic DNA extraction from post-mortem highly degraded skeletal remains. The analysis included 25 bone samples collected on autopsy. DNA extraction was performed in accordance with the QIAamp DNA Investigator Kit and PrepFiler Forensic DNA Extraction Kit manufacturer's isolation protocols. Amplification was performed on a Biometra termocycler using the AmpFISTR Identifiler PCR Amplification Kit according to the manufacturer's protocol. Typing of PCR products was carried out on an ABI Prism 377 DNA sequencer. The recommended parameters for GeneScan analysis and Genotyper software were followed. The authors demonstrated that the QIAamp DNA Investigator Kit was more effective, convenient and statistically significantly better method which may be employed in DNA extraction from bone specimens.

  7. Sensitivity of APTIMA HPV E6/E7 mRNA test in comparison with hybrid capture 2 HPV DNA test for detection of high risk oncogenic human papillomavirus in 396 biopsy confirmed cervical cancers.

    Science.gov (United States)

    Basu, Partha; Banerjee, Dipanwita; Mittal, Srabani; Dutta, Sankhadeep; Ghosh, Ishita; Chowdhury, Nilarun; Abraham, Priya; Chandna, Puneet; Ratnam, Sam

    2016-07-01

    The sensitivity of E6/E7 mRNA-based Aptima HPV test (AHPV; Hologic, Inc.) for detection of cervical cancer has been reported based on only a small number of cases. We determined the sensitivity of AHPV in comparison with the DNA-based Hybrid Capture 2 HPV test (HC2; Qiagen) for the detection of oncogenic HPV in a large number of cervical cancers at the time of diagnosis using cervical samples obtained in ThinPrep (Hologic). Samples yielding discordant results were genotyped using Linear Array assay (LA; Roche). Of 396 cases tested, AHPV detected 377 (sensitivity, 95.2%; 95%CI: 93.1-97.3), and HC2 376 (sensitivity, 94.9%; 95%CI: 92.7-97.1) with an agreement of 97.2% (kappa 0.7; 95%CI: 0.54-0.87). Among six AHPV+/HC2- cases, LA identified oncogenic HPV types in four including a type 73 and was negative in two. Among five AHPV-/HC2+ cases, LA detected oncogenic HPV types in two including a type 73 and was negative in three. Of 14 AHPV-/HC2- cases, 13 were genotyped. LA detected oncogenic HPV types in six, non-oncogenic types in three, and was negative in four. This is the largest study to demonstrate the sensitivity of AHPV for the detection of invasive cervical cancer and this assay showed equal sensitivity to HC2.

  8. Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities.

    Directory of Open Access Journals (Sweden)

    Hanna Johansson

    Full Text Available Continuous expression of E6- and E7-oncogenes of high-risk human papillomavirus (HPV types is necessary for the development and maintenance of the dysplastic phenotype. The aim of the study was to determine the sensitivity and specificity of the APTIMA HPV mRNA assay (Hologic in predicting future development of high-grade cervical intraepithelial neoplasia (CIN among high-risk HPV-DNA-positive women with atypical squamous cells of undetermined significance (ASCUS or low-grade squamous epithelial lesion (LSIL cytology.Archived SurePath cervical samples of women ≥ 35 years of age with high-risk HPV DNA-positive ASCUS (n = 211 or LSIL, (n = 131 were tested for the presence of high-risk HPV E6/E7 mRNA using the APTIMA HPV assay, and the women were monitored for development of histopathologically verified CIN2+.Twenty-nine percent (61/211 of the women in the ASCUS group, and 34.3% (45/131 in the LSIL group developed CIN2+ within 4.5 years of follow-up. The prevalence of HPV mRNA was 90.0% (95% CI 85.9-94.0 among women with ASCUS and 95.4% (95% CI 91.8-99.0 among women with LSIL. The presence of HPV E6/E7 mRNA was associated with future development of CIN2+ among women with ASCUS and LSIL (p=0.02. The mRNA assay demonstrated high sensitivity in predicting future CIN2+ and CIN3 for index ASCUS (96.7%; 95% CI 87.6-99.4 and 100%; 95% CI 82.2-100, respectively and LSIL (97.8%, 95% CI 86.8-99.9 and 100%, 95% CI 79.9-100, respectively. The corresponding specificity was low, 12.7% (95% CI 7.9-19.3 and 5.8% (95% CI 2.2-13.6, for future CIN2+, respectively. The negative predictive value of the HPV mRNA assay for detecting future CIN3 was 100%, since no mRNA-negative woman developed CIN3 (0/27 as compared to 13.6% (43/315 of the mRNA-positive women (p = 0.03.The APTIMA mRNA assay demonstrated high sensitivity but low specificity in predicting future CIN2+ among women with minor cytological abnormalities. The assay had high negative predictive value for future

  9. Diversity of HPV types in cancerous and pre-cancerous penile lesions of South African men: implications for future HPV vaccination strategies.

    Science.gov (United States)

    Lebelo, Ramokone L; Boulet, Gaëlle; Nkosi, Cornelius M; Bida, Mechack N; Bogers, John-Paul; Mphahlele, M Jeffrey

    2014-02-01

    This study reports the detection of HPV types from cancerous and pre-cancerous penile lesions that were diagnosed histologically. Sixty-six (22 pre-cancerous and 44 cancerous lesions) tissue biopsies, received between 2004 and 2011 by the Anatomical Pathology Department at Dr. George Mukhari Hospital were selected for this study. Total DNA was extracted and genotyped using type specific real-time quantitative polymerase chain reaction (qPCR) for 18 HPV types. Of 66 samples, only 51 were included in the analysis. Overall, HPV 11 (50.9%) and HPV 16 (49.1%) showed almost similar incidence in the study patients. In pre-cancerous lesions, HPV 11 was more frequent (80.0%), followed by HPV 31 and HPV 16 at 25.0% each and other HPV types included 35 (15.0%), 59 (15.0%), 53 (10.0%), 33 (10.0%), 18 (5.0%), 51 (5.0%), 52 (5.0%), 56 (5.0%), and 67 (5.0%). For cancerous lesions, HPV 16 was the most detected (62.9%), followed by HPV 11 (34.3%), and other HPV types included 18 (11.4%), 33 (5.7%), 39 (5.7%), 45 (5.7%), 66 (5.7%), 52 (2.9%), 58 (2.9%), 6 (2.9%), and 67 (2.9%). Several lesions demonstrated multiple HPV infections, ranging from two to six different types in one lesion. The study showed high diversity of HPV types in cancerous and pre-cancerous lesions of South African males with the most frequent being HPV types 11 and 16. The data suggest that boys could directly benefit from vaccination as they are exposed to variety of HPV types as early as 10 years of age in Africa.

  10. 深圳市妇女宫颈癌组织中人乳头瘤病毒16型E6、E7致癌基因的序列分析%Sequencing analysis of HPV16 E6/E7 gene extracted from cervical cancer tissues from female patient in Shenzhen

    Institute of Scientific and Technical Information of China (English)

    关嵩青; 林莉; 叶菲

    2014-01-01

    Objective To investigate the coding sequences of HPV1 6 E6/E7 gene extracted from the cervical cancer tissues from patients in Shenzhen.Methods The HPV1 6 E6/E7 gene was extracted from a HPV1 6 positive cervical cancer patient that had been living in Shenzhen for 1 7 years.The gene was amplified by PCR and cloned;the recombi-nant plasmid was constructed;and the DNA Sequence was restricted by restriction endonuclease.Results The HPV1 6 E6/E7 gene was successfully cloned and the sequencing analysis showed that the cloned HPV1 6 E6/E7 gene is identical to the German standard strain documented in GenBank.Conclusion The coding of HPV1 6 E6/E7 gene extracted from cervical cancer tissues from female patient in Shenzhen is identical to the German standard strain.%目的:检测深圳市宫颈癌患者癌组织中 HPV16型 E6、E7致癌基因的序列。方法采用 PCR 技术扩增1例在深圳生活17年宫颈癌 HPV16型阳性患者组织中 HPV16 E6、E7基因,对其进行克隆、构建重组质粒并进行核酸限制性内切酶鉴定及DNA测序分析。结果成功地克隆了 HPV16 E6、E7基因,测序分析表明克隆的 HPV16 E6、E7基因与 GenBank收录的德国标准株相比完全一致。结论深圳市妇女宫颈癌组织中 HPV16型 E6、E7致癌基因的序列与德国标准株相同。

  11. Intravaginal HPV DNA vaccination with electroporation induces local CD8+ T-cell immune responses and antitumor effects against cervicovaginal tumors.

    Science.gov (United States)

    Sun, Y; Peng, S; Qiu, J; Miao, J; Yang, B; Jeang, J; Hung, C-F; Wu, T-C

    2015-07-01

    Therapeutic human papillomavirus (HPV) vaccines have the potential to inhibit the progression of an established HPV infection to precancer and cancer lesions by targeting HPV oncoproteins. We have previously developed a therapeutic DNA vaccine encoding calreticulin (CRT) linked to E7, CRT/E7 DNA vaccine, for use in the treatment of HPV-associated lesions. Since the transfection efficiency of DNA vaccines administered in vivo is typically low, we examined the use of electroporation as well as different routes of administration to enhance antigen-specific tumor control. We tested the effects of the CRT/E7 DNA vaccine administered intramuscularly or intravaginally, with or without electroporation, on the generation of CD8+ T-cell immunity and therapeutic antitumor effects in HPV16 E7-expressing cervicovaginal tumor-bearing mice. We found that intravaginal vaccination of CRT/E7 DNA followed by electroporation-induced potent E7-specific CD8(+) T-cell responses in the cervicovaginal tract, compared with intramuscular injection followed by electroporation. Furthermore, tumor-bearing mice vaccinated intravaginally followed by electroporation had an enhanced survival, antitumor effects and local production of IFN-γ+CD8+ T cells compared with those vaccinated intramuscularly with electroporation. Thus, we show that intravaginal CRT/E7 DNA vaccination followed by electroporation generates the most potent therapeutic antitumor effects against an orthotopic E7-expressing tumor model. The current study will have significant clinical implications once a clinically applicable electroporation device for intravaginal use becomes available.

  12. Combined Analysis of HPV DNA and p16INK4a Expression to Predict Prognosis in ASCUS and LSIL Pap Smears

    OpenAIRE

    Carozzi, Francesca Maria

    2007-01-01

    Human papillomavirus (HPV) is known to play an important etiological role in the genesis of cervical cancer, but only a very small proportion of infected women develop invasive cervical cancer. The purpose of cervical cancer prevention is early diagnosis of its precursors. The molecular detection of HPV DNA as a diagnostic test to cervical carcinogenesis gave a low positive predictive value as compared to the use of biomarkers. p16INK4a has been proposed as putative surrogate biom...

  13. Evolution and taxonomic classification of human papillomavirus 16 (HPV16-related variant genomes: HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67.

    Directory of Open Access Journals (Sweden)

    Zigui Chen

    Full Text Available BACKGROUND: Human papillomavirus 16 (HPV16 species group (alpha-9 of the Alphapapillomavirus genus contains HPV16, HPV31, HPV33, HPV35, HPV52, HPV58 and HPV67. These HPVs account for 75% of invasive cervical cancers worldwide. Viral variants of these HPVs differ in evolutionary history and pathogenicity. Moreover, a comprehensive nomenclature system for HPV variants is lacking, limiting comparisons between studies. METHODS: DNA from cervical samples previously characterized for HPV type were obtained from multiple geographic regions to screen for novel variants. The complete 8 kb genomes of 120 variants representing the major and minor lineages of the HPV16-related alpha-9 HPV types were sequenced to capture maximum viral heterogeneity. Viral evolution was characterized by constructing phylogenic trees based on complete genomes using multiple algorithms. Maximal and viral region specific divergence was calculated by global and pairwise alignments. Variant lineages were classified and named using an alphanumeric system; the prototype genome was assigned to the A lineage for all types. RESULTS: The range of genome-genome sequence heterogeneity varied from 0.6% for HPV35 to 2.2% for HPV52 and included 1.4% for HPV31, 1.1% for HPV33, 1.7% for HPV58 and 1.1% for HPV67. Nucleotide differences of approximately 1.0% - 10.0% and 0.5%-1.0% of the complete genomes were used to define variant lineages and sublineages, respectively. Each gene/region differs in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1 /noncoding region 2 (NCR2 >upstream regulatory region (URR> E6/E7 > E2/L2 > E1/L1. CONCLUSIONS: These data define maximum viral genomic heterogeneity of HPV16-related alpha-9 HPV variants. The proposed nomenclature system facilitates the comparison of variants across epidemiological studies. Sequence diversity and phylogenies of this clinically important group of HPVs provides the basis for further studies of discrete

  14. Microscope Titration and Extraction of DNA from Liver.

    Science.gov (United States)

    Mayo, Lois T.; And Others

    1993-01-01

    Describes a simple and inexpensive, one-period activity to extract DNA to make the study of DNA less abstract. A microscope titration is used to determine when cells are ready for DNA extraction. (PR)

  15. Microscope Titration and Extraction of DNA from Liver.

    Science.gov (United States)

    Mayo, Lois T.; And Others

    1993-01-01

    Describes a simple and inexpensive, one-period activity to extract DNA to make the study of DNA less abstract. A microscope titration is used to determine when cells are ready for DNA extraction. (PR)

  16. The next-generation Hybrid Capture High-Risk HPV DNA assay on a fully automated platform.

    Science.gov (United States)

    Eder, Paul S; Lou, Jianrong; Huff, John; Macioszek, Jerzy

    2009-07-01

    A next-generation diagnostic system has been developed at QIAGEN. The QIAensemble system consists of an analytical subsystem (JE2000) that utilizes a re-engineered Hybrid Capture chemistry (NextGen) to maintain the high level of clinical sensitivity established by the digene High-Risk HPV DNA Test (HC2), while creating improved analytical specificity as shown both in plasmid-based analyses and in processing of clinical specimens. Limit-of-detection and cross-reactivity experiments were performed using plasmid DNA constructs containing multiple high-risk (HR) and low-risk (LR) HPV types. Cervical specimens collected into a novel specimen collection medium, DCM, were used to measure stability of specimens, as well as analytical specificity. Signal carryover, instrument precision, and specimen reproducibility were measured on the prototype JE2000 system using the automated NextGen assay. The Limit of Detection (LOD) is HPV 16 plasmid in the automated assay. No cross-reactivity (signal above cutoff) was detected on the automated system from any of 13 LR types tested at 10(7) copies per assay. Within-plate, plate-to-plate, and day-to-day performance in the prototype system yielded a CV of 20%. No indication of target carryover was found when samples containing up to 10(9) copies/ml of HPV DNA type 16 were processed on the JE2000 instrument. In an agreement study with HC2, 1038 donor cervical specimens were tested in both the manual NextGen assay and HC2 to evaluate agreement between the two tests. After eliminating discrepant specimens that were adjudicated by HR-HPV genotyping, the adjudicated positive agreement was 98.5% (95% CI: 94.6, 99.6). The JE2000 prototype system automates NextGen assay processing, yielding accurate, reproducible, and highly specific results with both plasmid analytical model tests and cervical specimens collected in DCM. The final system will process more than 2000 specimens in an 8-hour shift, with fully continuous loading.

  17. Status of carcinoma cervix and high risk HPV 16 DNA in women with postmenopausal uterine bleeding (PMB

    Directory of Open Access Journals (Sweden)

    Veena Kashyap

    2014-09-01

    Full Text Available Postmenopausal bleeding (PMB is a discharge that occurs following the firm diagnosis of menopause, which is at least six months from the end of women’s menstrual cycle but not to be confused with infrequent or irregular periods occurring around the time of menopause. It is a common problem representing 5% of all gynecology outpatient attendances which are to eliminate endometrial cancer as the cause of bleed and PMB should be reported urgently to the gynecologist. Uterine bleeding in postmenopausal women is highly indicative clinically of malignancy originating from cervix or endometrium and Human papilloma virus (HPV is one of the causative agent for carcinoma cervix. Incidence of carcinoma cervix increases with the age in mature women, however, incidence of human papillomavirus (HPV infection reduces as menopause sets in. The presence of the virus could be used as an early indication of disease potential. Because the Pap test can only detect clinical evidence of cervical disease, molecular-based diagnostic tools are being used more frequently to detect the virus before abnormal cell growth can be observed. This study was aimed to determine the status of cervical cancer and HPV 16 DNA positivity in relation to postmenopausal bleeding.

  18. Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods.

    Science.gov (United States)

    Chinami, M; Tanikawa, E; Hachisuka, H; Sasai, Y; Shingu, M

    1990-01-01

    The alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.

  19. Microbial DNA extraction from samples of varied origin

    National Research Council Canada - National Science Library

    S. Ray Chaudhuri; A. K. Pattanayak; A. R. Thakur

    2006-01-01

    The impact of four different soil DNA extraction methods on the quantity and quality of isolated community DNA was evaluated using agarose gel electrophoresis, DNA spectrum study and PCR-based 16S ribosomal DNA analysis...

  20. Design of a highly effective therapeutic HPV16 E6/E7-specific DNA vaccine: optimization by different ways of sequence rearrangements (shuffling.

    Directory of Open Access Journals (Sweden)

    Fahad N Almajhdi

    Full Text Available Persistent infection with the high-risk Human Papillomavirus type 16 (HPV 16 is the causative event for the development of cervical cancer and other malignant tumors of the anogenital tract and of the head and neck. Despite many attempts to develop therapeutic vaccines no candidate has entered late clinical trials. An interesting approach is a DNA based vaccine encompassing the nucleotide sequence of the E6 and E7 viral oncoproteins. Because both proteins are consistently expressed in HPV infected cells they represent excellent targets for immune therapy. Here we report the development of 8 DNA vaccine candidates consisting of differently rearranged HPV-16 E6 and E7 sequences within one molecule providing all naturally occurring epitopes but supposedly lacking transforming activity. The HPV sequences were fused to the J-domain and the SV40 enhancer in order to increase immune responses. We demonstrate that one out of the 8 vaccine candidates induces very strong cellular E6- and E7- specific cellular immune responses in mice and, as shown in regression experiments, efficiently controls growth of HPV 16 positive syngeneic tumors. This data demonstrates the potential of this vaccine candidate to control persistent HPV 16 infection that may lead to malignant disease. It also suggests that different sequence rearrangements influence the immunogenecity by an as yet unknown mechanism.

  1. HPV vaccine

    Science.gov (United States)

    ... cervix - HPV vaccine; Abnormal Pap smear - HPV vaccine; Vaccination - HPV vaccine ... and Gynecologists. Committee opinion No. 641: human papillomavirus vaccination. Obstet Gynecol . 2015;126(3):e38-e43. PMID: ...

  2. Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

    Science.gov (United States)

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-05-02

    Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS-protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol-chloroform extraction, are recommended. All of these steps are described in detail here.

  3. The diagnostic value of serum hybrid capture 2 (CH2) HPV DNA in cervical cancer: a systematic review and meta-analysis.

    Science.gov (United States)

    Yin, Duo; Jiang, Yan; Wang, Ning; Ouyang, Ling; Lu, Yanming; Zhang, Yao; Wei, Heng; Zhang, Shulan

    2014-09-01

    The diagnostic accuracy of cervical cancer remains a clinical challenge, and a number of studies have used the serum hybrid capture 2 (HC2) human papillomavirus (HPV) DNA in the diagnosis of cervical cancer. The aim of the present meta-analysis was to determine the overall accuracy of HC2 HPV DNA in the diagnosis of cervical cancer. A systematic review of studies from PubMed, Embase, the Cochrane Library, Web of Science, Ovid, Chinese Biomedical Literature Database-disc, Chinese National Knowledge Infrastructure (CNKI), Technology of Chongqing (VIP), and Wan Fang database was conducted, and the data concerning the accuracy of HC2 HPV DNA in the diagnosis of cervical cancer were pooled. The methodological quality of each study was assessed by quality assessment for studies of diagnostic accuracy (QUADAS). Statistical analysis was performed by employing Meta-DiSc (version 1.4) and Stata (version 12.0) software. The overall test performance was summarized using receiver operating characteristic curves. Finally, 12 studies, including 12,492 subjects, met the inclusion criteria and then included in this present meta-analysis. The summary estimates for serum HC2 HPV DNA in the diagnosis of cervical cancer were as follows: sensitivity 0.83 (95 % confidence interval (CI) 0.81-0.85), specificity 0.71 (95 % CI 0.69-0.72), positive likelihood ratio 3.65 (95 % CI 1.77-7.54), negative likelihood ratio 0.32 (95 % CI 0.21-0.48), and diagnostic odds ratio 10.54 (95 % CI 4.95-22.46), and the area under the curve was 0.8922. Our findings suggest that HC2 HPV DNA may improve the accuracy of cervical cancer diagnosis, while the results of HC2 HPV DNA assays should be interpreted in parallel with conventional test results and other clinical findings.

  4. p16INK4 expression in precursor lesions of squamous cell cervical cancer related to the presence of HPV-DNA

    Directory of Open Access Journals (Sweden)

    A.E.G. Godoy

    2008-07-01

    Full Text Available The purpose of the present study was to identify the expression of p16INK4 in cervical cancer precursor lesions by immunohistochemistry and to correlate it with lesion grade and presence of human papillomavirus (HPV infection. Cervical specimens from 144 women seen consecutively at the gynecology outpatient clinic of our institution from December 2003 to May 2005 were analyzed by cytopathology, histopathology, polymerase chain reaction for HPV-DNA, and p16INK4 immunostaining. Histologically normal biopsies, HPV-DNA negative by polymerase chain reaction, were used as control. HPV-DNA prevalence, including the control group, was 68.1% and the prevalence of p16INK4 expression was 55.0%. The percentage of cells stained by p16INK4 ranged from 10 to 100%, both in the group consisting of cervical intraepithelial neoplasia (CIN1/HPV specimens and in the group of CIN2/CIN3 specimens with P value of 0.0001. p16INK4 expression was 48.3% in the CIN1/HPV group, as opposed to 94.3% in the CIN2/CIN3 group (P = 0.001, showing a statistically significant difference between the two groups. The quantitative method used here is simple and less subjective than the different semiquantitative methods described in the literature. In view of the different definitions of a p16INK4-positive case, it is almost impossible to compare the findings reported by different investigators. This study confirms the association between p16INK4 and CIN2 and CIN3 lesions. Moreover, it shows that some low grade lesions expressed high levels of this protein. This may indicate that such low grade lesions may be predisposed to progress to high grade lesions. This means that p16INK4 may be a strong marker for "neoplastic lesions" induced by HPV and not just an infection marker.

  5. [Investigation on HPV DNA detection and genotyping practices used in French laboratories in 2009].

    Science.gov (United States)

    Heard, Isabelle; Favre, Michel; Fihman, Valentine

    2011-01-01

    The French National reference Laboratory for Human papillomavirus (HPV) performed in 2009 a national study in order to review the methods used to detect and identify HR HPV genotypes in microbiology laboratories. Results from this study show a great diversity in volumes of samples treated in laboratories. Among clinical indications, the most frequent is a result of ASC-US at a Pap smear. This indication in the only one covered by the National Public Insurance System and is mostly performed in laboratories from private sector. Other indications mainly correspond to research programs and are performed in public Hospitals. This study allowed also to review the adequacy between the liquid based cytology samples and the assays used for direct detection of HR HPV or identification of the genotypes present in the sample. The right tests were not carried in the right solution storage according to the recommendations from different HPV testing assays. National recommendations should be elaborated in order to improve the performance of the test used.

  6. Preclinical development of DNA vaccine candidates for the treatment of HPV16 induced malignancies

    NARCIS (Netherlands)

    Oosterhuis, Koen

    2012-01-01

    High-risk Human Papilloma Virus (HPV) induced malignancies are an interesting target for immunotherapy as they invariably express the viral proteins E6 and E7 that allow specific recognition of tumor cells without the risk of inducing autoimmunity. This thesis describes the preclinical development

  7. Molecular characterization of p16-immunopositive but HPV DNA-negative oropharyngeal carcinomas

    NARCIS (Netherlands)

    Rietbergen, M.M.; Snijders, P.J.F.; Beekzada, D.; Braakhuis, B.J.M.; Brink, A.; Heideman, D.A.M.; Hesselink, A.T.; Witte, B.I.; Bloemena, E.; Baatenburg-de Jong, R.J.; Leemans, C.R.; Brakenhoff, R.H.

    2014-01-01

    Recent studies have reported that p16 protein overexpression qualifies as a surrogate marker identifying an oncogenic human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC). However, there is still a percentage of OPSCCs that are positive for p16 immunohistochemistry (

  8. HPV and HPV Testing

    Science.gov (United States)

    ... does not directly affect the chances of getting pregnant. If HPV infection leads to cervical changes that need to be treated, the treatment should not affect your chances of getting pregnant. But if you have many treatments and biopsies, ...

  9. THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

    Institute of Scientific and Technical Information of China (English)

    Yang Jin; Li Xu; Li Ang; Wang Yili; Si Lüsheng

    2006-01-01

    Objective To construct eukaryotic expression vector of HPV18 L1- E6, E7 chimeric gene and examine the humoral and cellular immune responses induced by this DNA vaccines in mice. Methods The C-terminal of major capsid protein L1 gene and mutant zinc finger domains of early E6/7 oncogenes in HPV18 were integrated and inserted into eukaryotic expression vector pVAX1 to generate vaccines pVAX1-L1E6Mxx, E7Mxx. CHO cells were transiently transfected with the individual construct. Target protein expressions in the lysate of the transfected cells were measured by ELISA and immunocytochemistry. After BALB/c mice were vaccinated with various recombinant plasmids(pVAX1-L1-E6M3 or pVAX1-L1-E7M3) and immunie adjuvants (pLXHDmB7-2 or LTB) through different administration routes (intramuscular or intranasal) , the great cellular immune responses were produced as revealed by delayed-type hypersensitivity (DTH) and lymphocyte proliferation, and the expression of IL-4 and IFN- γ cells in CD4+ and CD8+subpopulations. Results The highly efficient expression of pVAX1-L1E6Mxx, E7Mxx vector in host eukaryotic cells were demonstrated both by ELISA and immunocytochemistry. The level of specific serum IgG against HPV in experiment groups mice was much higher than that of control group, and intranuscular immunization group had the highest antibody level. Intramuscular immunization groups were superior to intranasal immunization groups in DTH response, splenocyte proliferation and CD8+ IFN-γ + cells number, but CD4+ IL4+ cell number was higher in intranasal immunization groups. The immunization groups using pLXHDmB7-2 as adjuvant were superior to other groups in immunoresponse. Conclusion These DNA vaccines produce remarkable cellular and humoral immuneresponses in the mouse and may provide as prophylatic and therapeutic candidates for HPV induced cancer treatment.

  10. Extraction of ultrashort DNA molecules from herbarium specimens.

    Science.gov (United States)

    Gutaker, Rafal M; Reiter, Ella; Furtwängler, Anja; Schuenemann, Verena J; Burbano, Hernán A

    2017-02-01

    DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.

  11. An association analysis between mitochondrial DNA content, G10398A polymorphism, HPV infection, and the prognosis of cervical cancer in the Chinese Han population.

    Science.gov (United States)

    Feng, Dali; Xu, Hui; Li, Xin; Wei, Yuehua; Jiang, Huangang; Xu, Hong; Luo, Aihua; Zhou, Fuxiang

    2016-04-01

    The aim was to analyze quantitative (mitochondrial DNA (mtDNA) content) and qualitative (G10398A polymorphism) mtDNA alterations as well as human papillomavirus (HPV) infection in cervical cancer prognosis. One hundred and twenty-two cases of formalin-fixed paraffin-embedded cervical carcinoma specimens were collected from the Yichang Tumor Hospital and Zhongnan Hospital of Wuhan University in the recent 10 years together with medical records. A quantitative real-time PCR (RT-PCR) was used to determine the copy number of the mitochondrial DNA and HPV expression levels. G10398A polymorphism was determined by PCR-RFLP assay. The overall survival of patients with higher mtDNA content was significantly reduced compared with lower mtDNA content patients (P = 0.029). But there was no difference of prognosis between the mtDNA 10398 A allele and G allele. However, the Kaplan-Meier survival curve illustrated a significantly reduced overall survival in the patients with 10398A plus high mtDNA copy number compared with the other groups (P content compared with 10398G (P content were positively related in the younger subgroup (≤45 years) (correlation coefficient = 0.456, P = 0.022). This study indicated that mtDNA content and HPV infection status are associated with cervical cancer prognosis. High mitochondrial DNA content plus 10398 A may be a marker of poor prognosis in cervical cancer. And mtDNA variation may potentially influence the predisposition to HPV infection and cervical carcinogenesis.

  12. Methods for microbial DNA extraction from soil for PCR amplification

    Directory of Open Access Journals (Sweden)

    Yeates C

    1998-01-01

    Full Text Available Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1. DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

  13. Methods for microbial DNA extraction from soil for PCR amplification

    OpenAIRE

    Yeates C; Gillings, MR; Davison AD; Altavilla N; Veal DA

    1998-01-01

    Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol pr...

  14. Retrieval of HPV oncogenes E6 and E7 mRNA from cervical specimens using a manual open technology protocol.

    Science.gov (United States)

    Campbell, Leonardo Martins; Pitta, Denise Rocha; De Assis, Angela Maria; Derchain, Sophie Francoise Mauricette; Campos, Elisabete Aparecida; Sarian, Luis Otavio Zanatta

    2013-01-01

    HPV oncogenes mRNA detection gains momentum as an adjuvant for HPV-related cervical abnormalities diagnosis, but is based on costly detection assays not allowing viral type targeting. To assess detection rate of HPV oncogenes E6/E7 mRNA from cervical specimens using a manual, open technology, fully customizable protocol and determine whether HPV-related epidemiological features influence mRNA retrieval. We reviewed literature and compared our retrieval rate with automated technologies. We used 60 samples positive for HPV DNA types 16, 18, 31 and/or 45. We extracted mRNA with a TRizol-based protocol, and tested mRNA purity and concentration using light absorbance. We reverse-transcribed mRNA into cDNA for E6/7 detection. HPV oncogenes E6/E7 mRNA was retrieved from 36 (60%) out of 60 specimens. No HPV load-related clinical or epidemiological feature was significantly associated with mRNA retrieval. Presence of HPV-DNA 16/18 was associated with mRNA retrieval (OR = 9.08; 95% CI 1.26 to 65.32 for HPV 16; and 18.2; IC95% 1.86 to 391.44 for HPV 18). The open-technology protocol yielded an mRNA detection rate similar to that of automated technologies. Advantages are lower costs and target HPV type customization.

  15. 人乳头瘤病毒检测对宫颈病变的诊断价值%Clinical significance of the detection of the Human Papilloma Virus(HPV) DNA typing in therapy of cervical diseases

    Institute of Scientific and Technical Information of China (English)

    彭秀红; 毛玲芝; 罗喜平; 邓群娣; 林小红

    2009-01-01

    目的 评价人乳头瘤病毒(HPV-DNA)分型检测对宫颈病变的诊断价值.方法 对780例HPV-DNA阳性且病理活检为宫颈癌及宫颈上皮内瘤样病变(CIN Ⅰ-Ⅲ)患者进行手术或物理治疗,治疗后6个月、12个月行HPV-DNA检测及液基薄层细胞学检查.结果 780例患者中常见HPV-DNA分型分别为16、52、58、18、33、31型;在CIN Ⅰ患者中,HPV-52、58型检出率最高,在CIN Ⅱ、CIN Ⅲ、原位癌及浸润癌患者中,HPV-16型感染率明显高于其他基因型,差异有统计学意义(P<0.01).520例治疗后随访1年,其中432例(83.08%)HPV-DNA感染患者3个月至1年内消退,88例(16.92%)HPV感染未消退,其中细胞学检查为正常或炎症48例(54.55%),ASC-US14例(15.90%),LSIL 22例(25%),HSIL4例(4.55%);HPV持续阳性患者中细胞学检查异常均高于HPV阴性患者.结论 宫颈病变患者最常见HPV基因型为16、52、58、18、33、31型,尤其是HPV-16型与高级别上皮内瘤变及宫颈癌密切相关.%Objective To evaluate the clinical significance of the detection of the Human Papilloma Virus (HPV) DNA typing in therapy of cervical diseases. Methods 780 cases have been studied. These cases which showed HPV-DNA positive and were diagnosed by biopsy as cervical cancer or cervical intraepithelial neoplasia( CIN Ⅰ-Ⅲ) were treated by operation or physical therapy. And 6 months and 12 months after the treatment,TCT and HPV DNA test were carried out. Results The prevalence of HPV-DNA types among the 780 cases descended from HPV 16 to 52,58,18,33 and 31. Detection rates of HPV-52 and 58 were highest among the patients with CIN Ⅰ. In patients with CIN Ⅱ,CIN Ⅲ,carcinoma in situ and invasive carcinoma,the positive rate of HPV-16 was obviously higher than other genotypes,and the difference was significant (P<0. 01 ). 520 cases were followed-up after treatment,we found that HPV-DNA subsided within 3 months to 1 year in 432 cases. 88 cases still showed the HPV-DNA positive, among

  16. A Simplified Rice DNA Extraction Protocol for PCR Analysis

    Institute of Scientific and Technical Information of China (English)

    CHEN Wen-yue; CUI Hai-rui; BAO Jin-song; ZHOU Xiang-sheng; SHU Qing-yao

    2006-01-01

    A simple protocol was established for DNA extraction using etiolated rice seedlings, whereby rice DNA was directly extracted ir 0.5 mol/L NaOH solution in a single eppendorf tube. Results of comparative PCR analyses and electrophoresis showed that the DNA extracted using this method was as good and useful as that using standard CTAB method.

  17. Comparison of FFPE histological versus LBP cytological samples for HPV detection and typing in cervical cancer.

    Science.gov (United States)

    Kim, Geehyuk; Cho, Hyemi; Lee, Dongsup; Park, Sunyoung; Lee, Jiyoung; Wang, Hye-Young; Kim, Sunghyun; Park, Kwang Hwa; Lee, Hyeyoung

    2017-02-27

    Human papillomavirus (HPV) infection is closely associated with cervical cancer. This study analyzed HPV genotype prevalence in 75 cases of formalin-fixed paraffin embedded (FFPE) tissue samples from patients diagnosed with cervical cancer. Genotype prevalence was assessed using Reverse Blot Assay (REBA) and quantitative polymerase chain reaction (qPCR), which target the HPV L1 and HPV E6/E7 genes, respectively. HPV DNA chip tests were also performed using liquid based preparation (LBP) cytological samples from the same patients who provided the FFPE histological samples. We observed a slight difference in HPV genotype distribution as assessed by DNA chip versus REBA. One possible explanation for this difference is that normal regions could be mixed with lesion regions when cytological samples are extracted from each patient with cancer. For the detection of moderate dysplasia, the main target of diagnosis, this difference is anticipated to be greater. We also made several unexpected observations. For example, HPV multi-infection was not detected. Moreover, the rate of HPV positivity varied radically depending on the cancer origin, e.g. squamous cell carcinoma versus adenocarcinoma. Our results imply that it is important to determine whether cytological specimens are suitable for HPV genotyping analysis and cervical cancer diagnosis. Future research on the mechanisms underlying cervical cancer pathogenesis is also necessary.

  18. Study on the change of HPV DNA virus content, circulating blood and tissue miR indexes of patients with cervical cancer

    Institute of Scientific and Technical Information of China (English)

    Ru-Qiong Luo; LiangTang; Ping Xie; Yan-Mei Hu

    2016-01-01

    Objective:To study and observe the change status of HPV DNA virus content, circulating blood and tissue miR indexes of patients with cervical cancer.Methods:A total of 72 patients with cervical cancer in our hospital from February 2014 to September 2015 were selected as the observation group, 72 healthy women at the same period were selected as the control group, then the HPV DNA virus content and constitution, circulating blood and tissue miR indexes of two groups were analyzed and compared, and the detection results of observation group with stageⅠ,Ⅱ,Ⅲ andⅣ of cervical cancer were compared.Results:The rates of HPV DNA virus content and>500 in the observation group were all higher than those of control group, the circulating blood and tissue miR-21 were all higher than those of control group, while the circulating blood and tissue miR-126, miR-143 and miR-145 were all lower than those of control group, and the detection results of observation group with stageⅠ,Ⅱ,Ⅲ andⅣ of cervical cancer all had obvious differences, the differences were all significant.Conclusion:The changes of the status of HPV DNA virus content, circulating blood and tissue miR indexes of patients with cervical cancer are great, and the influence of cervical cancer stage for the detection results are great, so it has active guiding significance for the diagnosis and treatment of disease.

  19. Glycyrrhiza uralensis water extract enhances dendritic cell maturation and antitumor efficacy of HPV dendritic cell-based vaccine

    Science.gov (United States)

    Aipire, Adila; Li, Jinyu; Yuan, Pengfei; He, Jiang; Hu, Yelang; Liu, Lu; Feng, Xiaoli; Li, Yijie; Zhang, Fuchun; Yang, Jianhua; Li, Jinyao

    2017-01-01

    Licorice has been used as herbal medicine and natural sweetener. Here, we prepared Glycyrrhiza uralensis water extract (GUWE) and investigated the effect of GUWE on the maturation and function of dendritic cells (DCs) and its adjuvant effect on DC-based vaccine. We observed that GUWE dose-dependently promoted DC maturation and cytokine secretion through TLR4 signaling pathway. The capacity of DC to stimulate allogenic splenocyte proliferation was also enhanced by GUWE treatment. Compared with control group, GUWE treated DCs pulsed with human papillomavirus (HPV)-16 E6/E7 peptides significantly inhibited the tumor growth in both early and late therapeutic groups. In early therapeutic group, the frequencies of induced regulatory T cells (iTregs: CD4+CD25−Fopx3+) and CD4+ and CD8+ T cells were significantly decreased and increased, respectively. HPV-16-specific CD8+ T cell responses were significantly induced and negatively correlated with iTreg frequencies and tumor weight. These results indicated the immunoregulatory activities of licorice. PMID:28272545

  20. Glycyrrhiza uralensis water extract enhances dendritic cell maturation and antitumor efficacy of HPV dendritic cell-based vaccine.

    Science.gov (United States)

    Aipire, Adila; Li, Jinyu; Yuan, Pengfei; He, Jiang; Hu, Yelang; Liu, Lu; Feng, Xiaoli; Li, Yijie; Zhang, Fuchun; Yang, Jianhua; Li, Jinyao

    2017-03-08

    Licorice has been used as herbal medicine and natural sweetener. Here, we prepared Glycyrrhiza uralensis water extract (GUWE) and investigated the effect of GUWE on the maturation and function of dendritic cells (DCs) and its adjuvant effect on DC-based vaccine. We observed that GUWE dose-dependently promoted DC maturation and cytokine secretion through TLR4 signaling pathway. The capacity of DC to stimulate allogenic splenocyte proliferation was also enhanced by GUWE treatment. Compared with control group, GUWE treated DCs pulsed with human papillomavirus (HPV)-16 E6/E7 peptides significantly inhibited the tumor growth in both early and late therapeutic groups. In early therapeutic group, the frequencies of induced regulatory T cells (iTregs: CD4(+)CD25(-)Fopx3(+)) and CD4(+) and CD8(+) T cells were significantly decreased and increased, respectively. HPV-16-specific CD8(+) T cell responses were significantly induced and negatively correlated with iTreg frequencies and tumor weight. These results indicated the immunoregulatory activities of licorice.

  1. 宫颈液基细胞学与HPV-DNA在妇科普查中的作用%TCT and the combination of HPV-DNA in the census of women's diseases

    Institute of Scientific and Technical Information of China (English)

    罗业红; 沈琳菲; 邓廷坤; 刘莉

    2012-01-01

    Objective: to evaluate combine of the liquid-based cytology test and HPV-DNA test in checking gynecological diseases. Methods: This paper has selected 500 samples in all of the gynecological diseases tests form April 2009 to July 2012 randomly to Comparing the detection sensitivity and positive coincidence rate about liquid-based cytology test and HPV-DNA test. This paper adopts TBS grading criteria to comparing the results. The patients who cytological examination result were above ASCUS and HPV positive performed biopsy. Results: Cervical liquid-based cytology diagnostic results show:Normal range and benign reactive cell changing 407 cases (81.4%), HPV - DNA positive cytology diagnosis 106 cases (21.2%), and the pathological diagnosis positive cell 81 cases (16.2%). They are 54 cases confirmed for CIN-I、19 cases for CINII - III and 9 cases for SCC by biopsy. The result show it have huge difference between combine the liquid-based cytology test and HPV-DNA test and any method used alone. The samples are statistically significant (p<0.05).Conclusion: The application of TCT test combine with Higher-risk HPV test and TBS diagnostic criteria can improve the detection rate of cervical lesions.%目的评价液基细胞学检测和HPV-DNA检测的联合应用在妇女病普查中的作用.方法选取2009年4月-2012年07月在我院进行妇女病普查随机抽查的500例当成本文的研究对象,比较液基细胞学检测和HPV检测两种方法的检出敏感性及阳性符合率.诊断应用TBS分级标准,并对细胞学检查诊断为ASCUS以上者和HPV阳性者活检.结果宫颈液基细胞学诊断结果正常范围及良性反应性细胞改变407例(占81.4%),HPV-DNA阳性细胞学诊断106例(占21.2%),病理诊断结果阳性细胞81例(16.2%);经活检证实有54例为CIN-I,CINI-I I 19例,SCC 9例.单用TCT或HPV-DNA检测结果与联合检测比较差异显著,且具有统计学意义(P<0.05).结论 TCT检测结合高危型HPV检测技术的应

  2. Evaluation of a combined triple method to detect causative HPV in oral and oropharyngeal squamous cell carcinomas: p16 Immunohistochemistry, Consensus PCR HPV-DNA, and In Situ Hybridization

    Directory of Open Access Journals (Sweden)

    Pannone Giuseppe

    2012-02-01

    Full Text Available Abstract Background Recent emerging evidences identify Human Papillomavirus (HPV related Head and Neck squamous cell carcinomas (HN-SCCs as a separate subgroup among Head and Neck Cancers with different epidemiology, histopathological characteristics, therapeutic response to chemo-radiation treatment and clinical outcome. However, there is not a worldwide consensus on the methods to be used in clinical practice. The endpoint of this study was to demonstrate the reliability of a triple method which combines evaluation of: 1. p16 protein expression by immunohistochemistry (p16-IHC; 2. HPV-DNA genotyping by consensus HPV-DNA PCR methods (Consensus PCR; and 3 viral integration into the host by in situ hybridization method (ISH. This triple method has been applied to HN-SCC originated from oral cavity (OSCC and oropharynx (OPSCC, the two anatomical sites in which high risk (HR HPVs have been clearly implicated as etiologic factors. Methylation-Specific PCR (MSP was performed to study inactivation of p16-CDKN2a locus by epigenetic events. Reliability of multiple methods was measured by Kappa statistics. Results All the HN-SCCs confirmed HPV positive by PCR and/or ISH were also p16 positive by IHC, with the latter showing a very high level of sensitivity as single test (100% in both OSCC and OPSCC but lower specificity level (74% in OSCC and 93% in OPSCC. Concordance analysis between ISH and Consensus PCR showed a faint agreement in OPSCC (κ = 0.38 and a moderate agreement in OSCC (κ = 0.44. Furthermore, the addition of double positive score (ISHpositive and Consensus PCR positive increased significantly the specificity of HR-HPV detection on formalin-fixed paraffin embedded (FFPE samples (100% in OSCC and 78.5% in OPSCC, but reduced the sensitivity (33% in OSCC and 60% in OPSCC. The significant reduction of sensitivity by the double method was compensated by a very high sensitivity of p16-IHC detection in the triple approach. Conclusions Although HR

  3. Optimization of supercoiled HPV-16 E6/E7 plasmid DNA purification with arginine monolith using design of experiments.

    Science.gov (United States)

    Almeida, A M; Queiroz, J A; Sousa, F; Sousa, A

    2015-01-26

    The progress of DNA vaccines is dependent on the development of suitable chromatographic procedures to successfully purify genetic vectors, such as plasmid DNA. Human Papillomavirus is associated with the development of tumours due to the oncogenic power of E6 and E7 proteins, produced by this virus. The supercoiled HPV-16 E6/E7 plasmid-based vaccine was recently purified with the arginine monolith, with 100% of purity, but only 39% of recovery was achieved. Therefore, the present study describes the application of experimental design tools, a newly explored methodology in preparative chromatography, in order to improve the supercoiled plasmid DNA recovery with the arginine monolith, maintaining the high purity degree. In addition, the importance and influence of pH in the pDNA retention to the arginine ligand was also demonstrated. The Composite Central Face design was validated and the recovery of the target molecule was successfully improved from 39% to 83.5%, with an outstanding increase of more than double, while maintaining 100% of purity.

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

    DEFF Research Database (Denmark)

    Mirsepasi, Hengameh; Persson, Søren; Struve, Carsten

    2014-01-01

    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...... 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......) and a manual one (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were tested on stool samples collected from 3 patients with Inflammatory Bowel disease (IBD) and 5 healthy individuals. DNA extracts obtained by the QIAamp DNA Stool Mini Kit yield a higher amount of DNA compared to DNA extracts obtained...

  5. Advances in the researches of the therapeutic HPV DNA vaccines%治疗性HPV DNA疫苗的研究进展

    Institute of Scientific and Technical Information of China (English)

    徐思慧; 曹保利

    2015-01-01

    HPV感染与宫颈癌的发生密切相关.治疗性HPV DNA疫苗可以治疗HPV感染,进而阻断由HPV引起的宫颈癌前病变.基于HPV及DNA疫苗的深入研究,研究者通过不同方法来改进治疗性HPV DNA疫苗,以期在不久的将来能应用于临床.

  6. Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells.

    Science.gov (United States)

    Graham, Jessica C; Zarbl, Helmut

    2012-01-01

    Disease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is the approach used to select high affinity aptamers for specific macromolecular targets from among the >10(13) oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines. Whole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX)). Using this approach, we identified high affinity aptamers (nanomolar range K(d)) to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa). We also performed preliminary investigation of the aptamer epitopes and their binding characteristics. Using AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may be useful in predicting the tumorigenic potential and properties of cancer biopsies

  7. Use of cell-SELEX to generate DNA aptamers as molecular probes of HPV-associated cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Jessica C Graham

    Full Text Available BACKGROUND: Disease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX is the approach used to select high affinity aptamers for specific macromolecular targets from among the >10(13 oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines. METHODOLOGY/PRINCIPAL FINDINGS: Whole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX. Using this approach, we identified high affinity aptamers (nanomolar range K(d to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa. We also performed preliminary investigation of the aptamer epitopes and their binding characteristics. CONCLUSIONS/SIGNIFICANCE: Using AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may

  8. Efficacy of hybrid capture 2 HPV DNA testing on the screening of cervical cancer: a Meta-analysis%HPV DNA检测(HC2)用于宫颈癌筛查价值的Meta分析

    Institute of Scientific and Technical Information of China (English)

    李杨; 尚玉敏; 杨阳

    2013-01-01

    Objective:To evaluate the screening value of hybrid capture 2 (HC2) HPV DNA testing for cervical cancer by meta-analysis.Methods:By searching PubMed,Central(the Cochrane central register of controlled trials)、Embase、CBM、CNKI and WANFANG et al.All studies about HPV DNA test using HC2 for cervical cancer screening were retrieved from the data bases.The quality of included studies was evaluated by QUADAS items.The sensitivity,specificity and systematic receiver operating characteristic curves (SROC) were calculated to assess the screening value of individual diagnostic test.The data was analyzed by using statistic software Meta-Disc1.4 and STATA11.0.Results:Twelve cross-sectional studies that included a total of 12492 participants were analyzed in the current meta-analysis.The pooled values of sensitivity,specificity were 83% (95% CI 82% ~ 85%) and 88% (95% CI 87% ~ 89%) respectively.The area under SROC curve (AUC) of HPV DNA (HC2) array for cervical cancer screening was 0.91 (95%CI0.88 ~0.93).Condusion:HPV DNA test using HC2 has relative high sensitivity and specificity in screening of cervical cancer,which can be used as an useful method for cervical cancer screening.%目的:探讨2代杂交捕获法(HC2)检测HPV DNA在宫颈癌筛查中的应用价值.方法:计算机检索PubMed、Embase、Central、CNKI、CBMDisc、万方数据库等.纳入公开发表的有关HC2检测HPV DNA用于筛查宫颈癌的诊断试验,质量评价后,应用STATA11.0和Meta-discl.4软件进行Meta分析,评价HC2筛查宫颈癌的敏感性、特异性和ROC曲线下面积(AUC).结果:最终纳入12篇文献,共计12 492例.Meta分析结果显示,HC2检测HPV DNA筛查宫颈癌的敏感性和特异性分别为83%(95% CI为82%~85%)和88%(95%CI为87% ~89%);AUC为0.91(95% CI为0.88~0.93).结论:HC2检测HPV DNA筛查宫颈癌的敏感性和特异性均较高,可作为临床大规模宫颈癌筛查的有效方法.

  9. Optimized microbial DNA extraction from diarrheic stools

    Directory of Open Access Journals (Sweden)

    Donatin Emilie

    2012-12-01

    Full Text Available Abstract Background The detection of enteropathogens in stool specimens increasingly relies on the detection of specific nucleic acid sequences. We observed that such detection was hampered in diarrheic stool specimens and we set-up an improved protocol combining lyophilization of stools prior to a semi-automated DNA extraction. Findings A total of 41 human diarrheic stool specimens comprising of 35 specimens negative for enteropathogens and six specimens positive for Salmonella enterica in culture, were prospectively studied. One 1-mL aliquot of each specimen was lyophilised and total DNA was extracted from lyophilised and non-lyophilised aliquots by combining automatic and phenol-chloroform DNA extraction. DNA was incorporated into real-time PCRs targeting the 16S rRNA gene of Bacteria and the archaea Methanobrevibacter smithii and the chorismate synthase gene of S. enterica. Whereas negative controls consisting in DNA-free water remained negative, M. smithii was detected in 26/41 (63.4% non-lyophilised (Ct value 28.78 ± 9.1 versus 39/41 (95.1% lyophilised aliquots (Ct value 22.04 ± 5.5; bacterial 16S rRNA was detected in 33/41 (80.5% non-lyophilised (Ct value 28.11 ± 5.9 versus 40/41 (97.6% lyophilised aliquots (Ct value 24.94 ± 6.6; and S. enterica was detected in 6/6 (100% non-lyophilized and lyophilized aliquots (Ct value 26.98 ± 4.55 and 26.16 ± 4.97, respectively. S. enterica was not detected in the 35 remaining diarrheal-stool specimens. The proportion of positive specimens was significantly higher after lyophilization for the detection of M. smithii (p = 0.00043 and Bacteria (p = 0.015. Conclusion Lyophilization of diarrheic stool specimens significantly increases the PCR-based detection of microorganisms. The semi-automated protocol described here could be routinely used for the molecular diagnosis of infectious diarrhea.

  10. Successive DNA extractions improve characterization of soil microbial communities

    Directory of Open Access Journals (Sweden)

    Mauricio R. Dimitrov

    2017-02-01

    Full Text Available Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies describing and comparing soil microbial communities are based on a single DNA extraction, which may not lead to a representative recovery of DNA from all organisms present in the soil. The use of successive DNA extractions might improve soil microbial characterization, but the benefit of this approach has only been limitedly studied. To determine whether successive DNA extractions of the same soil sample would lead to different observations in terms of microbial abundance and community composition, we performed three successive extractions, with two widely used commercial kits, on a range of clay and sandy soils. Successive extractions increased DNA yield considerably (1–374%, as well as total bacterial and fungal abundances in most of the soil samples. Analysis of the 16S and 18S ribosomal RNA genes using 454-pyrosequencing, revealed that microbial community composition (taxonomic groups observed in the successive DNA extractions were similar. However, successive DNA extractions did reveal several additional microbial groups. For some soil samples, shifts in microbial community composition were observed, mainly due to shifts in relative abundance of a number of microbial groups. Our results highlight that performing successive DNA extractions optimize DNA yield, and can lead to a better picture of overall community composition.

  11. Purification of total DNA extracted from activated sludge

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible.

  12. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  13. Food Fish Identification from DNA Extraction through Sequence Analysis

    Science.gov (United States)

    Hallen-Adams, Heather E.

    2015-01-01

    This experiment exposed 3rd and 4th y undergraduates and graduate students taking a course in advanced food analysis to DNA extraction, polymerase chain reaction (PCR), and DNA sequence analysis. Students provided their own fish sample, purchased from local grocery stores, and the class as a whole extracted DNA, which was then subjected to PCR,…

  14. Genomic DNA extraction protocols from ovine hair

    Directory of Open Access Journals (Sweden)

    Jennifer Nonato da Silva Prate

    2013-12-01

    Full Text Available Genomic DNA extracted from animal cells can be used for several purposes, for example, to know genetic variability and genetic relationships between individuals, breeds and/or species, paternity tests, to describe the genetic profile for registration of the animal at association of breeders, detect genetic polymorphisms (SNP related to characteristics of commercial interest, disease diagnose, assess resistance or susceptibility to pathogens, etc. For such evaluations, in general, DNA is amplified by PCR (polymerase chain reaction, and then subjected to various techniques as RFLP (restriction fragments length polymorphism, SSCP (single strand conformation polymorphism, and sequencing. The DNA may be obtained from blood, buccal swabs, meat, cartilage or hair bulb. Among all, the last biological material has been preferred by farmers for its ease acquisition. Several methods for extracting DNA from hair bulb were reported without any consensus for its implementation. This study aimed to optimize a protocol for efficient DNA extraction for use in PCR-RFLP analysis of the Prion gene. For this study, were collected hair samples containing hair bulb from 131 Santa Inês sheep belonging to the Institute of Zootechny, Nova Odessa - SP. Two DNA extraction protocols were evaluated. The first, called phenol-chloroform-isoamyl alcohol (PCIA has long been used by Animal Genetic Laboratories, whose procedures are described below: in each microtube (1.5 mL containing 500 µL of TE-Tween solution (Tris-HCl 50 mM, EDTA 1 mM and 0.5% Tween 20 were added to approximately 30 hair bulb per animal which was incubated at 65°C with shaking at 170 rpm for 2 hours. Then was added 15 µL of proteinase K [10 mg mL-1] and incubated at 55°C at 170 rpm for 6-12 hours. At the end of digestion was added 1 volume of solution phenol-chloroform-isoamyl alcohol (25:24:1 followed by vigorous shaking for 10 seconds and centrifuged at 8000 rpm and 4°C for 10 minutes. The upper phase

  15. Leukocyte telomere length variation due to DNA extraction method.

    Science.gov (United States)

    Denham, Joshua; Marques, Francine Z; Charchar, Fadi J

    2014-12-04

    Telomere length is indicative of biological age. Shorter telomeres have been associated with several disease and health states. There are inconsistencies throughout the literature amongst relative telomere length measured by quantitative PCR (qPCR) and different extraction methods or kits used. We quantified whole-blood leukocyte telomere length using the telomere to single copy gene (T/S) ratio by qPCR in 20 young (18-25 yrs) men after extracting DNA using three common extraction methods: Lahiri and Nurnberger (high salt) method, PureLink Genomic DNA Mini kit (Life Technologies) and QiaAmp DNA Mini kit (Qiagen). Telomere length differences of DNA extracted from the three extraction methods was assessed by one-way analysis of variance (ANOVA). DNA purity differed between extraction methods used (P=0.01). Telomere length was impacted by the DNA extraction method used (P=0.01). Telomeres extracted using the Lahiri and Nurnberger method (mean T/S ratio: 2.43, range: 1.57-3.02) and PureLink Genomic DNA Mini Kit (mean T/S ratio: 2.57, range: 2.24-2.80) did not differ (P=0.13). Likewise, QiaAmp and Purelink-extracted telomeres were not statistically different (P=0.14). The Lahiri-extracted telomeres, however, were significantly shorter than those extracted using the QiaAmp DNA Mini Kit (mean T/S ratio: 2.71, range: 2.32-3.02; P=0.003). DNA purity was associated with telomere length. There are discrepancies between the length of leukocyte telomeres extracted from the same individuals according to the DNA extraction method used. DNA purity could be responsible for the discrepancy in telomere length but this will require validation studies. We recommend using the same DNA extraction kit when quantifying leukocyte telomere length by qPCR or when comparing different cohorts to avoid erroneous associations between telomere length and traits of interest.

  16. Development of optimal liquid based cytology sample processing methods for HPV testing: minimising the 'inadequate' test result.

    Science.gov (United States)

    Peevor, R; Jones, J; Fiander, A N; Hibbitts, S

    2011-05-01

    Incorporation of HPV testing into cervical screening is anticipated and robust methods for DNA extraction from liquid based cytology (LBC) samples are required. This study compared QIAamp extraction with Proteinase K digestion and developed methods to address DNA extraction failure (β-globin PCR negative) from clinical specimens. Proteinase K and QIAamp extraction methods in paired LBC samples were comparable with adequate DNA retrieved from 93.3% of clinical specimens. An HPV prevalence cohort (n=10,000) found 7% (n=676) LBC samples tested negative for β-globin, and were classified as inadequate. This 'failure' rate is unsuitable for population screening, particularly as the sampling method is intrusive. 379/676 samples were assessed to determine the cause of test failure. Re-testing confirmed adequate DNA in 21.6% of the original extracts; re-extraction from stored material identified 56.2% samples contained adequate material; dilution to overcome sample inhibition (1:10) resolved 51.7% cases in original extracts and 28% in new extracts. A standardised approach to HPV testing with an optimal DNA concentration input rather than standard volume input is recommended. Samples failing initial DNA extraction should be repeat extracted and assessed for sample inhibition to reduce the 7% of HPV tests being reported as inadequate and reduce the need for retesting of those women to <1%.

  17. Efficient genomic DNA extraction from low target concentration bacterial cultures using SCODA DNA extraction technology.

    Science.gov (United States)

    So, Austin; Pel, Joel; Rajan, Sweta; Marziali, Andre

    2010-10-01

    Methods for the extraction of nucleic acids are straightforward in instances where there is ample nucleic acid mass in the sample and contamination is minimal. However, applications in areas such as metagenomics, life science research, clinical research, and forensics, that are limited by smaller amounts of starting materials or more dilute samples, require sample preparation methods that are more efficient at extracting nucleic acids. Synchronous coefficient of drag alteration (SCODA) is a novel electrophoretic nucleic acid purification technology that has been tested successfully with both highly contaminated and dilute samples and is a promising candidate for new sample preparation challenges. In this article, as an example of SCODA's performance with limited sample material, we outline a genomic DNA (gDNA) extraction protocol from low abundance cultures of Escherichia coli DH10B. This method is equally well suited to high biomass samples.

  18. Overcoming DNA extraction problems from carnivorous plants

    Directory of Open Access Journals (Sweden)

    Fleischmann, Andreas

    2009-12-01

    Full Text Available We tested previously published protocols for DNA isolation from plants with high contents of polyphenols and polysaccharides for several taxa of carnivorous plants. However, we did not get satisfying results with fresh or silica dried leaf tissue obtained from field collected or greenhouse grown plants, nor from herbarium specimens. Therefore, we have developed a simple modified protocol of the commercially available Macherey- Nagel NucleoSpin® Plant kit for rapid, effective and reproducible isolation of high quality genomic DNA suitable for PCR reactions. DNA extraction can be conducted from both fresh and dried leaf tissue of various carnivorous plant taxa, irrespective of high contents of polysaccharides, phenolic compounds and other secondary plant metabolites that interfere with DNA isolation and amplification.

    Probamos algunos protocolos publicados previamente para el aislamiento del ADN de plantas con alto contenido de polifenoles y polisacáridos para varios táxones de plantas carnívoras. Sin embargo, no conseguimos muy buenos resultados ni con tejidos de hojas frescas, ni con tejidos de hojas secadas en gel de sílice obtenidas de plantas colectadas en el campo o cultivadas en los invernaderos, ni de especímenes de herbario. Por lo tanto, hemos desarrollado un protocolo sencillo, modificado del Macherey- Nagel NucleoSpin® Plant kit disponible en el mercado para el aislamiento rápido, eficaz y reproducible de ADN genómico de alta calidad conveniente para la reacción en cadena de la polimerasa. La extracción del ADN se puede realizar en tejidos de hojas frescas o secas de varios táxones de plantas carnívoras, sin importar el grado de contenido de polisacáridos, compuestos fenólicos u otros metabolitos secundarios que interfieren con el aislamiento y la amplificación del ADN.

  19. High quality genomic DNA extraction from postmortem fetal tissue.

    Science.gov (United States)

    Addison, S; Sebire, N J; Taylor, A M; Abrams, D; Peebles, D; Mein, C; Munroe, P B; Thayyil, S

    2012-11-01

    We examined the yield and quality of genomic deoxyribonucleic acid (DNA) extracted from various postmortem fetal tissues. Fetal tissues were collected at the time of autopsy, and DNA was subsequently extracted. The yield and DNA quality was assessed using ultraviolet spectrometry and agarose gel electrophoresis. We used polymerase chain reaction (PCR) to assess the DNA extracted for genomic testing. The median (range) gestation of the fetuses was 22 (16-41) weeks and the postmortem interval was 5.5 (2-10) days. Non-degraded genomic DNA was successfully extracted from all fetal tissues. Liver tissue had the lowest quality and muscle the highest quality. DNA yield or purity was not influenced by the postmortem interval. High quality genomic DNA can be extracted from fetal muscle, despite postmortem intervals of several days.

  20. 高危型HPV DNA检测在宫颈上皮内瘤变及宫颈早期鳞癌锥切术后随访中的应用及意义%Application and significance of high risk HPV(HR-HPV) DNA testing in the follow-ups after cold knife conization in cervical intraepithelial neoplasia (CIN) and micro invasive cervical squamous cell cancer

    Institute of Scientific and Technical Information of China (English)

    李岩; 崔竹梅; 娄艳辉; 刘丽芝; 唐蕊; 韩汶君

    2012-01-01

    Objective: To evaluate the application and significance of high risk HPV (HR-HPV) DNA detection in the follow-ups after cold knife conization (CKC)in cervical intra-epithelial neoplasia(CIN)and cervical squamous cell cancer of Stage IA1. Methods; A retrospective review was performed on 308 women who underwent of CINII ~ Ⅲ and cervical squamous cell cancer of Stage IA1 were treated by cold knife conization between Jan. 2008 to Oct. 2010 in our hospital. Then the patients were followed up by HR-HPV DNA testing(hybrid capture 2,HC2) and thin-layer liquid-based cytology (LCT) to observe the clearance of HR-HPV and recurrence of cervical lesions. The related factors of persistent infection of HR-HPV and persistent lesions or recurrence after CKC were analyzed. Results: By the end of follow-up, 10 patients were evidenced persistent lesions or recurrence. The persistent lesions or recurrence were more likely to occur when the HR-HPV infection persisted (P<0. 001). The pre-conization HR-HPV DNA high viral load and three- or four-quadrant disease involved were risk factors for predicting HR-HPV persistent infection after conization. HR-HPV DNA high viral load ( RIUs/COSs 500) was the only significantly independent risk factor for the persistent lesions or recurrence after conization. Conclusion: HR-HPV persistent infection after conization is a significantly important factor of the persistent lesions or recurrence. The pre-conization HR-HPV DNA high viral load is the only significantly independent risk factor of HR-HPV persistent infection and the persistent lesions or recurrence. Patients with high viral load prior to conization should be closely followed.%目的:探讨高危型HPV(HR-HPV)DNA检测在宫颈上皮内瘤变(GIN)及宫颈鳞癌(ⅠA1期)患者经宫颈冷刀锥切术治疗后随访中的应用及意义.方法:2008年1月至2010年10月我院收治的308例CINⅡ~Ⅲ及宫颈鳞癌(ⅠA1期)患者经宫颈冷刀锥切术治疗后,采

  1. Optimization of the Phenol -Chloroform Silica DNA Extraction Method in Ancient Bones DNA Extraction

    Directory of Open Access Journals (Sweden)

    Morteza Sadeghi

    2014-04-01

    Full Text Available Introduction: DNA extraction from the ancient bones tissues is currently very difficult. Phenol chloroform silica method is one of the methods currently used for this aim. The purpose of this study was to optimize the assessment method. Methods: DNA of 62 bone tissues (average 3-11 years was first extracted with phenol chloroform silica methods and then with changing of some parameters of the methods the extracted DNA was amplified in eight polymorphisms area including FES, F13, D13S317, D16, D5S818, vWA and CD4. Results from samples gained by two methods were compared in acrylamide gel. Results: The average of PCR yield for new method and common method in eight polymorphism regions was 75%, 78%, 81%, 76%, 85%, 71%, 89%, 86% and 64%, 39%, 70%, 49%, 68%, 76%, 71% and 28% respectively. The average of DNA in optimized (in 35l silica density and common method were 267.5 µg/ml with 1.12 purity and 192.76 g/ml with 0.84 purity respectively. Conclusions: According to the findings of this study, it is estimated that longer EDTA attendance is an efficient agent in removing calcium and also adequate density of silica particles can be efficient in removal of PCR inhibitors.

  2. Comparative analysis of protocols for DNA extraction from soybean caterpillars.

    Science.gov (United States)

    Palma, J; Valmorbida, I; da Costa, I F D; Guedes, J V C

    2016-04-07

    Genomic DNA extraction is crucial for molecular research, including diagnostic and genome characterization of different organisms. The aim of this study was to comparatively analyze protocols of DNA extraction based on cell lysis by sarcosyl, cetyltrimethylammonium bromide, and sodium dodecyl sulfate, and to determine the most efficient method applicable to soybean caterpillars. DNA was extracted from specimens of Chrysodeixis includens and Spodoptera eridania using the aforementioned three methods. DNA quantification was performed using spectrophotometry and high molecular weight DNA ladders. The purity of the extracted DNA was determined by calculating the A260/A280 ratio. Cost and time for each DNA extraction method were estimated and analyzed statistically. The amount of DNA extracted by these three methods was sufficient for PCR amplification. The sarcosyl method yielded DNA of higher purity, because it generated a clearer pellet without viscosity, and yielded high quality amplification products of the COI gene I. The sarcosyl method showed lower cost per extraction and did not differ from the other methods with respect to preparation times. Cell lysis by sarcosyl represents the best method for DNA extraction in terms of yield, quality, and cost effectiveness.

  3. Comparison of three DNA extraction methods for recovery of soil protist DNA.

    Science.gov (United States)

    Santos, Susana S; Nielsen, Tue Kjærgaard; Hansen, Lars H; Winding, Anne

    2015-08-01

    The use of molecular methods to investigate protist communities in soil is in rapid development this decade. Molecular analysis of soil protist communities is usually dependant on direct genomic DNA extraction from soil and inefficient or differential DNA extraction of protist DNA can lead to bias in downstream community analysis. Three commonly used soil DNA extraction methods have been tested on soil samples from three European Long-Term Observatories (LTOs) with different land-use and three protist cultures belonging to different phylogenetic groups in different growth stages. The methods tested were: ISOm-11063 (a version of the ISO-11063 method modified to include a FastPrep ®-24 mechanical lysis step), GnS-GII (developed by the GenoSol platform to extract soil DNA in large-scale soil surveys) and a commercial DNA extraction kit - Power Lyzer™ PowerSoil® DNA Isolation Kit (MoBio). DNA yield and quality were evaluated along with DNA suitability for amplification of 18S rDNA fragments by PCR. On soil samples, ISOm-11063 yields significantly higher DNA for two of the three soil samples, however, MoBio extraction favors DNA quality. This method was also more effective to recover copies of 18S rDNA numbers from all soil types. In addition and despite the lower yields, higher DNA quality was observed with DNA extracted from protist cultures with the MoBio method. Likewise, a bead-beating step shows to be a good solution for DNA extraction of soil protists, since the recovery of DNA from protist cultures and from the different soil samples with the ISOm method proved to be efficient in recovering PCR-amplifiable DNA. This study showed that soil DNA extraction methods provide biased results towards the cyst stages of protist organism. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Preliminary assessment for DNA extraction on microfluidic channel

    Science.gov (United States)

    Gopinath, Subash C. B.; Hashim, Uda; Uda, M. N. A.

    2017-03-01

    The aim of this research is to extract, purify and yield DNA in mushroom from solid state mushroom sample by using fabricated continuous high-capacity sample delivery microfluidic through integrated solid state extraction based amino-coated silica bead. This device is made to specifically extract DNA in mushroom sample in continuous inflow process with energy and cost consumption. In this project, we present two methods of DNA extraction and purification which are by using centrifuge (complex and conventional method) and by using microfluidic biosensor (new and fast method). DNA extracted can be determined by using ultraviolet-visible spectroscopy (UV-VIS). The peak obtained at wavelength 260nm after measuring the absorbance of sample proves that DNA is successfully extracted from the mushroom.

  5. A comparison of DNA extraction methods using Petunia hybrida tissues.

    Science.gov (United States)

    Tamari, Farshad; Hinkley, Craig S; Ramprashad, Naderia

    2013-09-01

    Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium bromide and chloroform-isoamyl alcohol and the Edwards method that uses the anionic detergent SDS and isopropyl alcohol. Our results show that the Edwards method works better than the CTAB method for extracting DNA from tissues of Petunia hybrida. For six of the eight tissues, the Edwards method yielded more DNA than the CTAB method. In four of the tissues, this difference was statistically significant, and the Edwards method yielded 27-80% more DNA than the CTAB method. Among the different tissues tested, we found that buds, 4 days before anthesis, had the highest DNA concentrations and that buds and reproductive tissue, in general, yielded higher DNA concentrations than other tissues. In addition, DNA extracted using the Edwards method was more consistently PCR-amplified than that of CTAB-extracted DNA. Based on these results, we recommend using the Edwards method to extract DNA from plant tissues and to use buds and reproductive structures for highest DNA yields.

  6. Further Stimulation of Cellular Immune Responses through Association of HPV-16 E6, E7 and L1 Genes in order to produce more Effective Therapeutic DNA Vaccines in Cervical Cancer Model.

    Science.gov (United States)

    Fazeli, Maryam; Soleimanjahi, Hoorieh; Dadashzadeh, Simin

    2015-01-01

    Cervical cancer has been shown to be highly associated with human papillomavirus (HPV) infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore potent targets for therapeutic genetic vaccination. In the present study, it was investigated the potential effect of HPV-16 E6, E7 and L1 co-administration to activate specific cytotoxic T lymphocytes in tumor mice models. The HPV-16 E6, E7 and L1 genes from Iranian isolate were separately inserted into the mammalian expression vector, pcDNA3, to construct the DNA vaccine candidates. Tumor-bearing Animals (C57BL/6 mice) were immunized with the vaccine candidate; then, Lymphocyte Proliferation Assay (LPA) and relative tumor volume measurements were carried out in order to examine the immunological effects of the vaccine. Obtained results showed that co-administration of the HPV-16 E6, E7 and L1 DNA induced HPV-16 specific cellular immune responses and also protected against TC-1-induced tumor in vivo compared with negative controls. The results showed that mixed delivery systems might be valuable to improve the magnitude of the induced immune responses and confirmed therapeutic effects of HPV-16 E6, E7 through cytotoxic T lymphocyte induction and illustrate the new promising role for HPV-16 L1 CTL epitopes as a suitable CTL inducer.

  7. Correlation between HPV sperm infection and male infertility

    Institute of Scientific and Technical Information of China (English)

    Yang Yang; Chan-Wei Jia; Yan-Min Ma; Li-Ying Zhou; Shu-Yu Wang

    2013-01-01

    Human papillomavirus (HPV) is one of the most common sexually transmitted diseases which comprises a group of small DNA viruses that intect both cutaneous and mucous squamous epithelia.Liquid bead microarray technology (LBMA) were used to evaluate 24 HPV genotypes in confirmed fertile and infertile males of North China so that the effects of HPV infection on semen parameters and relationship with male infertility could be discussed.A total of 1138 subjects were recruited in this study; 142 were HPV-pesitive (12.48%).Among 523 confirmed fertile males,only 35 were HPV-positive (6.70%),and two of them had multiple infections.Among 615 infertile males,107 were HPV-positive (17.4%),and 29 of them had multiple infections.Infertile males had a relatively high HPV infection rate compared with confirmed fertile males.Sperm progpessive motility (PR) and the normal morphology rate were significantly decreased in HPV-positive subjects.HPV-45,HPV-52,HPV-18,HPV-59 and HPV-16 infections were more frequently in infertile males.Hence,HPV infection is closely related to male infertility which will decrease sperm PR and rnorphology.HPV-45,HPV-52,HPV-18,HPV-59 and HPV-16 infection seems to be major risk factors.

  8. HPV16E7-HSP70 Hybrid DNA Vaccine Induces E7-Specific Cytotoxic T Cells and Antitumor Immunity

    Institute of Scientific and Technical Information of China (English)

    ZHU Liqin; LI Hui; XIONG Jinhu; WANG Tongxiang; OU Xuan; WEI Yun; WU Xinxing

    2006-01-01

    Using human papillomavirus type 16 (HPV16) E7 as an antigen and Heat Shock Protein 70 as adjuvant, we constructed a DNA vaccine by linking HSP70 gene to E7C91G gene. Mice, after being immunized with E7C91G-HSP70, E7C91G/HSP70, E7C91G, and wild E7 DNA vaccines respectively, produced E7 specific CD8+ T-cell precursor frequencies oF280. 33±2.52, 144.34±4. 04, 164.34±5.13 and 82.33± 3.51 respectively within every 1 × 105 mouse splenocytes. This proves that E7C91G-HSP70 fusion vaccine can significantly enhance the E7 specific cellular immunity within the mice body(p<0.01). After being immunized with E7C91G-HSP70 fusion vaccine, tumor-bearing mice of the group being treated have significantly longer latency and survival periods, comparing with other three categories of E7 vaccines. Experiment shows that this vaccine has a significant effect on enhancing E7 positive tumor-treatment within mice body. After being immunized with E7C91G-HSP70 vaccine, there were no pathological changes found in livers, kidneys and spleens of the mice, which proves that the vaccine is quite safe. After all,E7C91G-HSP70 fusion vaccine has a much stronger tumor- treatment effect than that of wild type E7 DNA vaccine.

  9. Evaluation of 14 triage strategies for HPV DNA-positive women in population-based cervical screening

    NARCIS (Netherlands)

    Rijkaart, Dorien C.; Berkhof, Johannes; van Kemenade, Folkert J.; Coupe, Veerle M. H.; Hesselink, Albertus T.; Rozendaal, Lawrence; Heideman, Danielle A. M.; Verheijen, Ren H.; Bulk, Saskia; Verweij, Wim M.; Snijders, Peter J. F.; Meijer, Chris J. L. M.

    2012-01-01

    High-risk human papillomavirus (hrHPV) testing has a higher sensitivity but lower specificity than cytology for detection of high-grade intraepithelial neoplasia (CIN). To avoid over-referral to colposcopy and overtreatment, hrHPV-positive women require triage testing and/or followup. A total of 25,

  10. [DNA extraction methods of compost for molecular ecology analysis].

    Science.gov (United States)

    Yang, Zhao-Hui; Xiao, Yong; Zeng, Guang-Ming; Liu, Yun-Guo; Deng, Jiu-Hua

    2006-08-01

    Molecular ecology provides new techniques for studying compost microbes, and the DNA extraction is the basis of molecular techniques. Because of the contamination of humic acids, it turns to be more difficult for compost microbial DNA extraction. Three different approaches, named as lysozyme lysis, ultrasonic lysis and proteinase K lysis with CTAB, were used to extract the total DNA from compost. The detection performed on a nucleic acids and protein analyzer showed that all the three approaches produced high DNA yields. The agarose gel electrophoresis showed that the DNA fragments extracted from compost had a length of about 23 kb. A eubacterial 16S rRNA gene targeted primer pair (27F and 1 495R) was used for PCR amplification, and all the samples got almost the full length 16S rDNA sequence (about 1.5 kb). After digested by restriction endonucleases (Hae Ill and Alu I), the restriction map showed relatively identical microbial diversity in the DNA, which was extracted by the three different approaches. All the compost microbial DNA extracted by the three different approaches could be used for molecular ecological study, and researchers should choose the right approach for extracting microbial DNA from compost based on the facts.

  11. Successive DNA extractions improve characterization of soil microbial communities

    NARCIS (Netherlands)

    Dimitrov, M.R.; Veraart, A.J.; De Hollander, M.; Smidt, H.; van Veen, J.A.; Kuramae, E.E.

    2017-01-01

    Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies describing

  12. Successive DNA extractions improve characterization of soil microbial communities

    NARCIS (Netherlands)

    Rocha Dimitrov, Mauricio; Veraart, Annelies J.; Hollander, de Mattias; Smidt, Hauke; Veen, van Johannes A.; Kuramae, Eiko E.

    2017-01-01

    Currently, characterization of soil microbial communities relies heavily on the use of molecular approaches. Independently of the approach used, soil DNA extraction is a crucial step, and success of downstream procedures will depend on how well DNA extraction was performed. Often, studies

  13. An efficient method for DNA extraction from Cladosporioid fungi

    NARCIS (Netherlands)

    Moslem, M.A.; Bahkali, A.H.; Abd-Elsalam, K.A.; Wit, de P.J.G.M.

    2010-01-01

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on

  14. An efficient method for DNA extraction from Cladosporioid fungi

    NARCIS (Netherlands)

    Moslem, M.A.; Bahkali, A.H.; Abd-Elsalam, K.A.; Wit, de P.J.G.M.

    2010-01-01

    We developed an efficient method for DNA extraction from Cladosporioid fungi, which are important fungal plant pathogens. The cell wall of Cladosporioid fungi is often melanized, which makes it difficult to extract DNA from their cells. In order to overcome this we grew these fungi for three days on

  15. Genital Warts (HPV)

    Science.gov (United States)

    ... A Week of Healthy Breakfasts Shyness Genital Warts (HPV) KidsHealth > For Teens > Genital Warts (HPV) A A ... HPV infection. How Do People Know They Have HPV? Most HPV infections have no signs or symptoms. ...

  16. Protocol for extraction of genomic DNA from swine solid tissues

    Directory of Open Access Journals (Sweden)

    Fernando Henrique Biase

    2002-01-01

    Full Text Available Molecular diagnostics are performed by using DNA from different body tissues. However, it is necessary to obtain genomic DNA of good quality. Due to the impossibility of collecting blood from slaughtered animals, DNA extraction from solid tissues is necessary. The objective of this study was to describe a protocol of DNA extraction from swine skin, adipose, brain, liver, kidney and muscle tissues. We obtained high molecular weight DNA of good quality, shown by agarose gel and amplification of two DNA fragments, 605bp and 891pb, by PCR. Spectrophotometric analysis of DNA concentration showed variation among the DNA from different tissues, with the liver and adipose tissues presenting the greatest and the smallest concentration, respectively. The described protocol has proven to be advantageous due to its simplicity, quickness, affordable reagents and absence of phenol, resulting in a high molecular weight DNA of good quality from several tissues.

  17. The First Attested Extraction of Ancient DNA in Legumes (Fabaceae).

    Science.gov (United States)

    Mikić, Aleksandar M

    2015-01-01

    Ancient DNA (aDNA) is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae) are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analyzing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum) and bitter vetch (Vicia ervilia) from Hissar, southeast Serbia, dated to 1,350-1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB) method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl(-1) of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK, and rbcL) among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighboring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide.

  18. Establishment of the one-step DNA extraction method of human papillomavirus in cervical epithelium samples%一步法宫颈脱落细胞人乳头瘤病毒 DNA 抽提方法的建立

    Institute of Scientific and Technical Information of China (English)

    赵琪

    2014-01-01

    Objective:To develop an one-step method on DNA extraction of human papillomavirus in cervical epithelium samples.Methods:The extraction of HPV-DNA in 200 cases collected from cervical epithelium samples was treated by column method of DNA extraction kit or one-step method.DNA purity and concentration of the samples were measured by the trace nucleic acid spectrophotometer.HPV geno-types were detected by PCR reverse dot blot(PCR-RDB)method.Results:DNA purity extracted by col-umn method and one-step were (1 .91 ±0.1 4)μg/mL and (1 .86 ±0.1 9)μg/mL,respectively,and con-centration of the HPV-DNA were (1 39.78 ±1 8.21 )μg/mL and (1 24.36 ±1 7.35)μg/mL,respectively. DNA purity and concentration extracted by two methods were all not statistically significant(P <0.01 ).The rates of positive,single infection and multiple infection of HPV genotyping treated by the column method and one-step method were 1 7.5%(35 /200),7.5%(1 5 /200),1 0%(20 /200)and 1 7.5%(35 /200),8%(1 6 /200),9.5%(1 9 /200),respectively.The value of Kappa was 0.886,which indicated that there was a high degree of consistency.Conclusion:The one-step method on HPV DNA extraction was simpler,the DNA quality was high and suitable for HPV DNA genotyping of the cervical epithelium samples.%目的:建立一步法抽提宫颈脱落细胞人乳头瘤病毒(human papillomavirus,HPV)DNA 的方法。方法:收集200例宫颈脱落细胞标本,分别采用柱式法 DNA 提取试剂盒和一步法抽提 HPV-DNA,用微量核酸测定仪测定 DNA 的纯度和浓度,采用 PCR 反向斑点杂交(PCR-RDB)法进行 HPV-DNA 分型检测。结果:柱式法抽提的 DNA 纯度和浓度分别为(1.91±0.14)μg/mL 和(139.78±18.21)μg/mL,一步法抽提的 DNA 纯度和浓度分别为(1.86±0.19)μg/mL和(124.36±17.35)μg/mL,两种方法的 DNA 纯度和浓度差异均有统计学意义(P <0.01)。柱式法和一步法 HPV 分型的总阳性率、

  19. A RAPID PCR-QUALITY DNA EXTRACTION METHOD IN FISH

    Institute of Scientific and Technical Information of China (English)

    LI Zhong; LIANG Hong-Wei; ZOU Gui-Wei

    2012-01-01

    PCR has been a general preferred method for biological research in fish, and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories[1-4]. The same problem among these procedures is waiting for tissue digesting for a long time. The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay, especially in large-scale PCR amplification, such as SSR-based genetic-mapping construction [5,6], identification of germ plasm resource[7,8] and evolution research [9,10], etc. In this study, a stable and rapid PCR-quality DNA extraction method was explored, using a modified alkaline lysis protocol. Extracting DNA for PCR only takes approximately 25 minutes. This stable and rapid DNA extraction method could save much laboratory time and promotes.%PCR has been a general preferred method for biological research in fish,and previous research have enabled us to extract and purify PCR-quality DNA templates in laboratories [1-4].The same problem among these procedures is waiting for tissue digesting for a long time.The overabundance time spent on PCR-quality DNA extraction restricts the efficiency of PCR assay,especially in large-scale PCR amplification,such as SSR-based genetic-mapping construction [5,6],identification of germ plasm resource[7,8] and evolution research [9,10],etc.In this study,a stable and rapid PCR-quality DNA extraction method was explored,using a modified alkaline lysis protocol.Extracting DNA for PCR only takes approximately 25 minutes.This stable and rapid DNA extraction method could save much laboratory time and promotes.

  20. Evaluation of six commercial DNA extraction kits for recovery of Burkholderia pseudomallei DNA.

    Science.gov (United States)

    Marques, Maria Angela de Mello; Zimmermann, Pia; Messelhäußer, Ute; Sing, Andreas

    2012-12-01

    Six commercially available DNA extraction kits, as well as thermal lysis and proteinase K DNA extraction were evaluated regarding bacterial inactivation, DNA yield and purity, and their use in a Burkholderia pseudomallei real-time PCR. While all methods successfully inactivated the bacteria, by measuring DNA purity and the level of detection by real-time PCR, the proteinase K method was the most sensitive.

  1. The positivity for human papillomavirus (HPV DNA and evaluation of probable risk factors among women in Eskisehir region

    Directory of Open Access Journals (Sweden)

    Ferhat Gurkan Aslan

    2015-06-01

    Conclusions: In addition to the prevention of cervical cancer, scanning programs are also important in protection against HPV infection. Applications which could be implemented on this subject can be considered as defining the potential risk factors and taking precautions against them. More detailed studies should be conducted in Turkey to research the relationship between HPV infections and social groups, the frequency of HPV infection,results, follow-up, treatment, control and prevention. Communities must be made aware of this subject throughpublic health education. [TAF Prev Med Bull 2015; 14(3.000: 222-228

  2. THE HUMORAL AND CELLULAR IMMUNE RESPONSES INDUCED BY HPV18L1-E6/E7 DNA VACCINES IN MICE

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Cervical cancer is the second most commoncancer in woman,particularly in developing coun-tries.In the past t wo decades,epidemiologic andvirological data have identified a clear and consistentassociation of human papilomavirus(HPV)infec-tion withthe development of cervical cancer[1].Theexpression of the E6and E7genes from high-riskHPV16and HPV18is crucial for development,i m-mortalization and maintenance of the malignantphenotype of cervical carcinoma.Therefore,E6andE7genes are i mportant targets for the de...

  3. High incidence of HPV-associated head and neck cancers in FA deficient mice is associated with E7's induction of DNA damage through its inactivation of pocket proteins.

    Directory of Open Access Journals (Sweden)

    Jung Wook Park

    Full Text Available Fanconi anemia (FA patients are highly susceptible to solid tumors at multiple anatomical sites including head and neck region. A subset of head and neck cancers (HNCs is associated with 'high-risk' HPVs, particularly HPV16. However, the correlation between HPV oncogenes and cancers in FA patients is still unclear. We previously learned that FA deficiency in mice predisposes HPV16 E7 transgenic mice to HNCs. To address HPV16 E6's oncogenic potential under FA deficiency in HNCs, we utilized HPV16 E6-transgenic mice (K14E6 and HPV16 E6/E7-bi-transgenic mice (K14E6E7 on genetic backgrounds sufficient or deficient for one of the fanc genes, fancD2 and monitored their susceptibility to HNCs. K14E6 mice failed to develop tumor. However, E6 and fancD2-deficiency accelerated E7-driven tumor development in K14E6E7 mice. The increased tumor incidence was more correlated with E7-driven DNA damage than proliferation. We also found that deficiency of pocket proteins, pRb, p107, and p130 that are well-established targets of E7, could recapitulate E7's induction of DNA damage. Our findings support the hypothesis that E7 induces HPV-associated HNCs by promoting DNA damage through the inactivation of pocket proteins, which explains why a deficiency in DNA damage repair would increase susceptibility to E7-driven cancer. Our results further demonstrate the unexpected finding that FA deficiency does not predispose E6 transgenic mice to HNCs, indicating a specificity in the synergy between FA deficiency and HPV oncogenes in causing HNCs.

  4. HPV Test

    Science.gov (United States)

    ... I'm a woman who has had the HPV vaccine. Do I need cervical cancer screening? Yes. Because currently available HPV vaccines do ... 2014. Accessed July 13, 2015. World Health Organization. Human Papillomavirus and Cervical Cancer. Available online at http://www.who.int/mediacentre/ ...

  5. HPV Vaccine

    Science.gov (United States)

    ... Surgery? A Week of Healthy Breakfasts Shyness HPV Vaccine KidsHealth > For Teens > HPV Vaccine Print A A A What's in this article? ... 11 or 12 through age 21 If needed, kids can get the vaccine starting at age 9. continue How Does the ...

  6. The influence of substrate on DNA transfer and extraction efficiency.

    Science.gov (United States)

    Verdon, Timothy J; Mitchell, R John; van Oorschot, Roland A H

    2013-01-01

    The circumstances surrounding deposition of DNA profiles are increasingly becoming an issue in court proceedings, especially whether or not the deposit was made by primary transfer. In order to improve the currently problematic evaluation of transfer scenarios in court proceedings, we examined the influence a variety of nine substrate types (six varieties of fabric, plywood, tarpaulin, and plastic sheets) has on DNA transfer involving blood. DNA transfer percentages were significantly higher (p=0.03) when the primary substrate was of non-porous material (such as tarpaulin, plastic or, to a lesser degree, wood) and the secondary substrate porous (such as fabrics). These findings on transfer percentages confirm the results of previous studies. Fabric composition was also shown to have a significant (p=0.03) effect on DNA transfer; when experiments were performed with friction from a variety of fabrics to a specific weave of cotton, transfer percentages ranged from 4% (flannelette) to 94% (acetate). The propensity for the same nine substrates to impact upon the efficiency of DNA extraction procedures was also examined. Significant (p=0.03) differences were found among the extraction efficiencies from different materials. When 15μL of blood was deposited on each of the substrates, the lowest quantity of DNA was extracted from plastic (20ng) and the highest quantities extracted from calico and flannelette (650ng). Significant (pDNA extraction yield from different initial blood volumes from all substrates. Also, significantly greater (pDNA was seen during concentration of extracts with higher compared to lower initial quantities of DNA. These findings suggest that the efficiency of extraction and concentration impacts upon the final amount of DNA available for analysis and that consideration of these effects should not be ignored. The application of correction factors to adjust for any variation among extraction and concentration efficiencies among substrates is

  7. Cervical HPV Infection in Female Sex Workers: A Global Perspective

    OpenAIRE

    Soohoo, Melissa; Blas, Magaly; Byraiah, Gita; Carcamo, Cesar; Brown, Brandon

    2013-01-01

    Introduction: Approximately 291 million women worldwide are HPV DNA carriers. Studies have indicated that having multiple sexual partners may lead to higher HPV transmission. Thus female sex workers (FSWs) may be at greater risk of infection compared to the general population. Herein we review publications with data on FSW cervical HPV test results. We also examine variations of HPV prevalence and risk behaviors by region. Knowledge of prevalent HPV types in FSWs may lead to improved preventi...

  8. The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 Positive and apoptosis in HeLa (HPV-18 positive.

    Directory of Open Access Journals (Sweden)

    Amit S Choudhari

    Full Text Available Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive and HeLa (HPV-18 positive cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca(2+ leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.

  9. Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

    Science.gov (United States)

    Atanesyan, Lilit; Steenkamer, Maryvonne J; Horstman, Anja; Moelans, Cathy B; Schouten, Jan P; Savola, Suvi P

    2017-01-01

    Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

  10. Extracting DNA of nematodes communities from Argentine Pampas agricultural soils

    Directory of Open Access Journals (Sweden)

    Eduardo A. Mondino

    2015-06-01

    Full Text Available We examined four strategies (Tris/EDTA, sodium dodecyl sulfate, Chelex 100 resin and cetyltrimethylammonium bromide -CTAB- for extracting nucleic acid (DNA from communities of nematodes. Nematodes were isolated from an agricultural area under different management of long-term crop rotation experiment from Argentina during three seasons. After DNA extraction, Polymerase Chain Reaction-amplifications were performed and considered as indicators of successful DNA extraction. The CTAB combined with proteinase K and phenol-chloroform-isoamyl alcohol was the unique successful method because positive amplifications were obtained by using both eukaryotic and nematode specific primers. This work could contribute to biodiversity studies of nematodes on agroecosystems.

  11. Extracting DNA of nematodes communities from Argentine Pampas agricultural soils.

    Science.gov (United States)

    Mondino, Eduardo A; Covacevich, Fernanda; Studdert, Guillermo A; Pimentel, João P; Berbara, Ricardo L L

    2015-01-01

    We examined four strategies (Tris/EDTA, sodium dodecyl sulfate, Chelex 100 resin and cetyltrimethylammonium bromide -CTAB-) for extracting nucleic acid (DNA) from communities of nematodes. Nematodes were isolated from an agricultural area under different management of long-term crop rotation experiment from Argentina during three seasons. After DNA extraction, Polymerase Chain Reaction-amplifications were performed and considered as indicators of successful DNA extraction. The CTAB combined with proteinase K and phenol-chloroform-isoamyl alcohol was the unique successful method because positive amplifications were obtained by using both eukaryotic and nematode specific primers. This work could contribute to biodiversity studies of nematodes on agroecosystems.

  12. The Need for Cervical Cancer Control in HIV-Positive and HIV-Negative Women from Romania by Primary Prevention and by Early Detection Using Clinically Validated HPV/DNA Tests.

    Directory of Open Access Journals (Sweden)

    Ramona Gabriela Ursu

    Full Text Available In Romania, a country with no organized national surveillance program regarding cervical cancer, the early diagnosis of HPV (Human Papilloma Virus infections is a major requirement, especially in HIV-infected women. The objective of this study was to determine the HPV prevalence and type distribution in young HIV-positive women and to assess the difference in the risk factors for developing cervical cancer compared to those of HIV-negative women.We conducted one cross-sectional cohort study from June 2013-September 2014, including 1,032 women: 992 HIV- women who were 36.5 years old (limits: 17 ÷ 84 and 40 HIV + women who were 22.9 years old (limits: 17 ÷ 30 with iatrogenic HIV infected. We detected HPV types with the Linear Array HPV Genotyping test (Roche, Romania.DNA/HPV was detected in 18/40 (45% of the HIV+ patients and in 350/992 (35.2% of the HIV- patients (OR = 1.5, 95%CI 0.76÷2.96. After age adjustment, the overall HPV prevalence was 51.6% in HIV+ versus 63.2% in HIV- women aged under 25, and 22.2% in HPV+ versus 47.2% in HIV- women aged 25-34. We detect HIV being a risk factor for acquiring multiple HPV type infections (OR = 2.30, 95% CI 0.88÷5.97. The eight most common HPV types (high-risk, and low-risk for women below age 30, HIV+ / - were: HPV 16, 18, 31, 51, 58, 68, and 6 and 82 respectively. To assess the risk factors of HIV-positive women for acquiring HPV infection, we analyzed the CD4/μL, ARN/HIV copies/μL, the age group, the number of sexual partners, smoking, and the type of HPV infection (single versus multiple infections. We found that the number of sexual partners and smoking are statistically significant risk factors.Even though there are no significant differences regarding the prevalence of HPV infection in HIV + versus HIV - patients, multiple infections were more frequent in the first group. In our study group young HIV-infected patients under HAART therapy, high number of sexual partners (more than 3 and smoking

  13. Extraction of high quality DNA from bloodstains using diatoms.

    Science.gov (United States)

    Günther, S; Herold, J; Patzelt, D

    1995-01-01

    A simple method is described for the extraction of high quality DNA for PCR amplification. The DNA was extracted by using Chelex-100 ion exchange resin or a special cell lysis buffer containing proteinase K. For further purification the DNA was bound to silica in the presence of a chaotrophic agent. Hence it is possible to unlimitedly wash the bound DNA and inhibitory substances are removed. By using diatoms as a source of silicates, this method is very economical and can therefore be used as a routine method.

  14. A rapid and efficient assay for extracting DNA from fungi

    Science.gov (United States)

    Griffin, Dale W.; Kellogg, C.A.; Peak, K.K.; Shinn, E.A.

    2002-01-01

    Aims: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. Methods and Results: Tissue (DNA for PCR/ sequencing applications. Conclusions: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. Significance and Impact of the Study: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.

  15. Necessity of Purification during Bacterial DNA Extraction with Environmental Soils.

    Science.gov (United States)

    Lim, Hyun Jeong; Choi, Jung-Hyun; Son, Ahjeong

    2017-08-08

    Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for PCR assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium) showed that sand samples containing less than 10 ug/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of magnesium ion was different from other inhibitors due to the complexation interaction of magnesium ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 ug/g of humic acids, less than 70% clay content and less than 0.01% magnesium ion content.

  16. Extraction of DNA from plant and fungus tissues in situ

    Directory of Open Access Journals (Sweden)

    Abu Almakarem Amal S

    2012-06-01

    Full Text Available Abstract Background When samples are collected in the field and transported to the lab, degradation of the nucleic acids contained in the samples is frequently observed. Immediate extraction and precipitation of the nucleic acids reduces degradation to a minimum, thus preserving accurate sequence information. An extraction method to obtain high quality DNA in field studies is described. Findings DNA extracted immediately after sampling was compared to DNA extracted after allowing the sampled tissues to air dry at 21°C for 48 or 72 hours. While DNA extracted from fresh tissues exhibited little degradation, DNA extracted from all tissues exposed to 21°C air for 48 or 72 hours exhibited varying degrees of degradation. Yield was higher for extractions from fresh tissues in most cases. Four microcentrifuges were compared for DNA yield: one standard electric laboratory microcentrifuge (max rcf = 16,000×g, two battery-operated microcentrifuges (max rcf = 5,000 and 3,000 ×g, and one manually-operated microcentrifuge (max rcf = 120×g. Yields for all centrifuges were similar. DNA extracted under simulated field conditions was similar in yield and quality to DNA extracted in the laboratory using the same equipment. Conclusions This CTAB (cetyltrimethylammonium bromide DNA extraction method employs battery-operated and manually-operated equipment to isolate high quality DNA in the field. The method was tested on plant and fungus tissues, and may be adapted for other types of organisms. The method produced high quality DNA in laboratory tests and under simulated field conditions. The field extraction method should prove useful for working in remote sites, where ice, dry ice, and liquid nitrogen are unavailable; where degradation is likely to occur due to the long distances between the sample site and the laboratory; and in instances where other DNA preservation and transportation methods have been unsuccessful. It may be possible to adapt

  17. An efficient genomic DNA extraction from whole blood using Nextractor.

    Science.gov (United States)

    Jeong, Tae-Dong; Cho, Young-Uk; Lee, Woochang; Chun, Sail; Min, Won-Ki

    2014-08-05

    We evaluated the performance of the Nextractor NX-48 nucleic acid extractor system for the extraction of genomic DNA from whole blood samples. We compared the performance of the Nextractor to that of the QIAamp DNA Blood Mini Kit and the Maxwell system, using five whole blood samples. Extraction efficiencies were compared based on the total amount of extracted DNA adjusted by input blood volume, and the purity was compared. Polymerase chain reaction analyses were performed using ACTB as a target. The real-time PCR assay was carried out for housekeeping gene GAPDH. Total elapsed time for DNA extraction was compared. Extraction efficiencies for the QIAamp, Maxwell, and Nextractor were 25.4±3.8ng/μL, 9.2±0.6ng/μL, and 31.0±5.6ng/μL, respectively. No significant differences in purity were observed among three methods. DNA extracted using the ACTB was successfully amplified in all three methods. There were no obvious differences in Ct values for GAPDH real-time PCR. Total elapsed time for DNA extraction was about 50min for the QIAamp, 40min for the Maxwell, and 20min for the Nextractor. As the Nextractor is faster and requires less hands-on time than manual procedures, it may be useful for molecular diagnostic testing in clinical laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Rapid extraction and preservation of genomic DNA from human samples.

    Science.gov (United States)

    Kalyanasundaram, D; Kim, J-H; Yeo, W-H; Oh, K; Lee, K-H; Kim, M-H; Ryew, S-M; Ahn, S-G; Gao, D; Cangelosi, G A; Chung, J-H

    2013-02-01

    Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

  19. A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples.

    Science.gov (United States)

    Claassen, Shantelle; du Toit, Elloise; Kaba, Mamadou; Moodley, Clinton; Zar, Heather J; Nicol, Mark P

    2013-08-01

    Differences in the composition of the gut microbiota have been associated with a range of diseases using culture-independent methods. Reliable extraction of nucleic acid is a key step in identifying the composition of the faecal microbiota. Five widely used commercial deoxyribonucleic acid (DNA) extraction kits (QIAsymphony® Virus/Bacteria Midi Kit (kit QS), ZR Fecal DNA MiniPrep™ (kit Z), QIAamp® DNA Stool Mini Kit (kit QA), Ultraclean® Fecal DNA Isolation Kit (kit U) and PowerSoil® DNA Isolation Kit (kit P)) were evaluated, using human faecal samples. Yield, purity and integrity of total genomic DNA were compared spectrophotometrically and using gel electrophoresis. Three bacteria, commonly found in human faeces were quantified using real time polymerase chain reaction (qPCR) and total bacterial diversity was studied using denaturing gradient gel electrophoresis (DGGE) as well as terminal restriction fragment length polymorphism (T-RFLP). The measurements of DNA yield and purity exhibited variations between the five kits tested in this study. Automated kit QS exhibited the best quality and highest quantity of DNA. All kits were shown to be reproducible with CV values≤0.46 for DNA extraction. qPCR results showed that all kits were uniformly efficient for extracting DNA from the selected target bacteria. DGGE and T-RFLP produced the highest diversity scores for DNA extracted using kit Z (H'=2.30 and 1.27) and kit QS (H'=2.16 and 0.94), which also extracted the highest DNA yields compared to the other kits assessed. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Extraction of DNA by magnetic ionic liquids: tunable solvents for rapid and selective DNA analysis.

    Science.gov (United States)

    Clark, Kevin D; Nacham, Omprakash; Yu, Honglian; Li, Tianhao; Yamsek, Melissa M; Ronning, Donald R; Anderson, Jared L

    2015-02-03

    DNA extraction represents a significant bottleneck in nucleic acid analysis. In this study, hydrophobic magnetic ionic liquids (MILs) were synthesized and employed as solvents for the rapid and efficient extraction of DNA from aqueous solution. The DNA-enriched microdroplets were manipulated by application of a magnetic field. The three MILs examined in this study exhibited unique DNA extraction capabilities when applied toward a variety of DNA samples and matrices. High extraction efficiencies were obtained for smaller single-stranded and double-stranded DNA using the benzyltrioctylammonium bromotrichloroferrate(III) ([(C8)3BnN(+)][FeCl3Br(-)]) MIL, while the dicationic 1,12-di(3-hexadecylbenzimidazolium)dodecane bis[(trifluoromethyl)sulfonyl]imide bromotrichloroferrate(III) ([(C16BnIM)2C12(2+)][NTf2(-), FeCl3Br(-)]) MIL produced higher extraction efficiencies for larger DNA molecules. The MIL-based method was also employed for the extraction of DNA from a complex matrix containing albumin, revealing a competitive extraction behavior for the trihexyl(tetradecyl)phosphonium tetrachloroferrate(III) ([P6,6,6,14(+)][FeCl4(-)]) MIL in contrast to the [(C8)3BnN(+)][FeCl3Br(-)] MIL, which resulted in significantly less coextraction of albumin. The MIL-DNA method was employed for the extraction of plasmid DNA from bacterial cell lysate. DNA of sufficient quality and quantity for polymerase chain reaction (PCR) amplification was recovered from the MIL extraction phase, demonstrating the feasibility of MIL-based DNA sample preparation prior to downstream analysis.

  1. A Novel Method of Genomic DNA Extraction for Cactaceae

    Directory of Open Access Journals (Sweden)

    Shannon D. Fehlberg

    2013-03-01

    Full Text Available Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies.

  2. Comparison of four methods of DNA extraction from rice

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ Polyphenols, teroens, and resins make it difficult to obtain high quality genomic DNA from rice. Four extraction methods were compared in our study, and CTAB precipitation was the most practical one.

  3. A novel method of genomic DNA extraction for Cactaceae1

    Science.gov (United States)

    Fehlberg, Shannon D.; Allen, Jessica M.; Church, Kathleen

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and complete avoidance of mucilage as compared to typical tissue extractions. Sequences from one nuclear and three plastid regions were obtained across eight genera and 20 species of cacti using DNA extracted from spines. • Conclusions: Genomic DNA useful for amplification and sequencing can be obtained from cactus spines. The protocols described here are valuable for any cactus species, but are particularly useful for investigators interested in sampling living collections, extensive field sampling, and/or conservation genetic studies. PMID:25202521

  4. Influence of EDTA and magnesium on DNA extraction from blood ...

    African Journals Online (AJOL)

    Influence of EDTA and magnesium on DNA extraction from blood samples and specificity of polymerase chain reaction. ... African Journal of Biotechnology ... of initial EDTA level added to blood samples on quantity and quality of genomic DNA ...

  5. DNA extraction for streamlined metagenomics of diverse environmental samples.

    Science.gov (United States)

    Marotz, Clarisse; Amir, Amnon; Humphrey, Greg; Gaffney, James; Gogul, Grant; Knight, Rob

    2017-06-01

    A major bottleneck for metagenomic sequencing is rapid and efficient DNA extraction. Here, we compare the extraction efficiencies of three magnetic bead-based platforms (KingFisher, epMotion, and Tecan) to a standardized column-based extraction platform across a variety of sample types, including feces, oral, skin, soil, and water. Replicate sample plates were extracted and prepared for 16S rRNA gene amplicon sequencing in parallel to assess extraction bias and DNA quality. The data demonstrate that any effect of extraction method on sequencing results was small compared with the variability across samples; however, the KingFisher platform produced the largest number of high-quality reads in the shortest amount of time. Based on these results, we have identified an extraction pipeline that dramatically reduces sample processing time without sacrificing bacterial taxonomic or abundance information.

  6. Prevalence and Genotype Distribution of HPV Infection in China: Analysis of 51,345 HPV Genotyping Results from China's Largest CAP Certified Laboratory.

    Science.gov (United States)

    Zeng, Zhengyu; Yang, Huaitao; Li, Zaibo; He, Xuekui; Griffith, Christopher C; Chen, Xiamen; Guo, Xiaolei; Zheng, Baowen; Wu, Shangwei; Zhao, Chengquan

    2016-01-01

    The prevalence of cervical Human Papillomavirus (HPV) infection varies greatly worldwide and data regarding HPV prevalence and genotypes in China are limited. HPV testing results were retrospectively examined at KingMed Diagnostics, the largest independent pathology laboratory in China, from January 2011 to June 2014. All testing was performed using the 26 HPV Genotyping Panel of Tellgenplex (TM) xMAP™ HPV DNA Test assay (TELLGEN, Shanghai, China). Overall prevalence, age-specific prevalence and genotype distributions were analyzed. A total of 51,345 samples were tested and the overall HPV prevalence was 26%, with 21.12% positive for high risk (HR) HPV and 8.37% positive for low risk HPV. 80% of HPV positive cases were positive for a single HPV type. The three most common HR HPV types detected were HPV-52, -16, and -58, in descending order. HPV-18 was only the 6(th) most common type. When women were divided into three age groups: HR HPV had the highest prevalence rate in women HR HPV genotypes also varied among these three age groups. To the best of our knowledge, this is largest routine clinical practice report of HPV prevalence and genotypes in a population of women having limited cervical cancer screening. HPV-52 was the most prevalent HR HPV type in this population of women followed by HPV-16 and HPV-58. The overall and age-specific prevalence and genotype distribution of HR HPV are different in this Chinese population compared to that reported from Western countries.

  7. A Novel Method of Genomic DNA Extraction for Cactaceae

    OpenAIRE

    Fehlberg, Shannon D.; Jessica M. Allen; Kathleen Church

    2013-01-01

    • Premise of the study: Genetic studies of Cactaceae can at times be impeded by difficult sampling logistics and/or high mucilage content in tissues. Simplifying sampling and DNA isolation through the use of cactus spines has not previously been investigated. • Methods and Results: Several protocols for extracting DNA from spines were tested and modified to maximize yield, amplification, and sequencing. Sampling of and extraction from spines resulted in a simplified protocol overall and compl...

  8. A simplified universal genomic DNA extraction protocol suitable for PCR.

    Science.gov (United States)

    Wang, T Y; Wang, L; Zhang, J H; Dong, W H

    2011-03-29

    Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 μg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.

  9. Optimization of DNA extraction for advancing coral microbiota investigations.

    Science.gov (United States)

    Weber, Laura; DeForce, Emelia; Apprill, Amy

    2017-02-08

    DNA-based sequencing approaches are commonly used to identify microorganisms and their genes and document trends in microbial community diversity in environmental samples. However, extraction of microbial DNA from complex environmental samples like corals can be technically challenging, and extraction methods may impart biases on microbial community structure. We designed a two-phase study in order to propose a comprehensive and efficient method for DNA extraction from microbial cells present in corals and investigate if extraction method influences microbial community composition. During phase I, total DNA was extracted from seven coral species in a replicated experimental design using four different MO BIO Laboratories, Inc., DNA Isolation kits: PowerSoil®, PowerPlant® Pro, PowerBiofilm®, and UltraClean® Tissue & Cells (with three homogenization permutations). Technical performance of the treatments was evaluated using DNA yield and amplification efficiency of small subunit ribosomal RNA (SSU ribosomal RNA (rRNA)) genes. During phase II, potential extraction biases were examined via microbial community analysis of SSU rRNA gene sequences amplified from the most successful DNA extraction treatments. In phase I of the study, the PowerSoil® and PowerPlant® Pro extracts contained low DNA concentrations, amplified poorly, and were not investigated further. Extracts from PowerBiofilm® and UltraClean® Tissue and Cells permutations were further investigated in phase II, and analysis of sequences demonstrated that overall microbial community composition was dictated by coral species and not extraction treatment. Finer pairwise comparisons of sequences obtained from Orbicella faveolata, Orbicella annularis, and Acropora humilis corals revealed subtle differences in community composition between the treatments; PowerBiofilm®-associated sequences generally had higher microbial richness and the highest coverage of dominant microbial groups in comparison to the Ultra

  10. Home-based urinary HPV DNA testing in women who do not attend cervical cancer screening clinics.

    Science.gov (United States)

    Ducancelle, Alexandra; Reiser, Justine; Pivert, Adeline; Le Guillou-Guillemette, Hélène; Le Duc-Banaszuk, Anne Sophie; Lunel-Fabiani, Françoise

    2015-09-01

    In France, cervical cancer screening is currently based on cytological examination of a Pap smear for women aged 25 to 65, but screening coverage is unsatisfactory. Previous studies have shown that self-sampling for human papillomavirus (HPV) testing increases rates of compliance. With this purpose in mind, we performed the CapU study to evaluate the acceptance of a urinary HPV test. Letters proposing a new cervical cancer screening method using at-home urine self-sampling were sent to 5000 women aged 40-65 years who had not had a Pap smear over the past three years. The participating patients had to send their urine samples to the Angers Hospital Virology Laboratory for analysis using real-time PCR. Of the 771 samples received, 687 were analyzed. High-risk HPV were detected in 29 women. In follow-up, 28 women with positive urinary HPV results had a Pap smear or colposcopy done. The cytological results showed nine abnormal Pap smears, among which histology studies confirmed three cases of cervical intraepithelial neoplasia grade III lesions. Our study shows that urinary HPV testing may be pertinent to women who do not have cervical Pap smears done and lead to the diagnosis of high-grade cervical lesions.

  11. Rapid Extraction of Human DNA Containing Humic Acid

    OpenAIRE

    Sutlović, Davorka; Definis Gojanović, Marija; Anđelinović, Šimun

    2007-01-01

    The identification process of dead bodies or human remains is nowadays conducted in numerous fields of forensic science, archeology and other judicial cases. A particular problem is the isolation and DNA typing of human remains found in mass graves, due to the degradation process, as well as post mortal DNA contamination with bacteria, fungi, humic acids, metals, etc. In this study, the influence of humic acid (HA) on the DNA extraction and typing is investigated. If present in...

  12. A Comparison of DNA Extraction Methods using Petunia hybrida Tissues

    OpenAIRE

    Tamari, Farshad; Hinkley, Craig S.; Ramprashad, Naderia

    2013-01-01

    Extraction of DNA from plant tissue is often problematic, as many plants contain high levels of secondary metabolites that can interfere with downstream applications, such as the PCR. Removal of these secondary metabolites usually requires further purification of the DNA using organic solvents or other toxic substances. In this study, we have compared two methods of DNA purification: the cetyltrimethylammonium bromide (CTAB) method that uses the ionic detergent hexadecyltrimethylammonium brom...

  13. Extracting biological knowledge from DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    De La Vega, F.M. [CINVESTAV-IPN (Mexico); Thieffry, D. [Universite Libre de Bruxelles, Rhode-Saint-Genese (Belgium)]|[Universidad Nacional Autonoma de Mexico, Morelos (Mexico); Collado-Vides, J. [Universidad Nacional Autonoma de Mexico, Morelos (Mexico)

    1996-12-31

    This session describes the elucidation of information from dna sequences and what challenges computational biologists face in their task of summarizing and deciphering the human genome. Techniques discussed include methods from statistics, information theory, artificial intelligence and linguistics. 1 ref.

  14. The first attested extraction of ancient DNA in legumes (Fabaceae

    Directory of Open Access Journals (Sweden)

    Aleksandar M. Mikić

    2015-11-01

    Full Text Available Ancient DNA (aDNA is any DNA extracted from ancient specimens, important for diverse evolutionary researches. The major obstacles in aDNA studies are mutations, contamination and fragmentation. Its studies may be crucial for crop history if integrated with human aDNA research and historical linguistics, both general and relating to agriculture. Legumes (Fabaceae are one of the richest end economically most important plant families, not only from Neolithic onwards, since they were used as food by Neanderthals and Paleolithic modern man. The idea of extracting and analysing legume aDNA was considered beneficial for both basic science and applied research, with an emphasis on genetic resources and plant breeding. The first reported successful and attested extraction of the legume aDNA was done from the sample of charred seeds of pea (Pisum sativum and bitter vetch (Vicia ervilia from Hissar, southeast Serbia, dated to 1,350 - 1,000 Before Christ. A modified version of cetyltrimethylammonium bromide (CTAB method and the commercial kit for DNA extraction QIAGEN DNAesy yielded several ng μl-1 of aDNA of both species and, after the whole genome amplification and with a fragment of nuclear ribosomal DNA gene 26S rDNA, resulted in the detection of the aDNA among the PCR products. A comparative analysis of four informative chloroplast DNA regions (trnSG, trnK, matK and rbcL among the modern wild and cultivated pea taxa demonstrated not only that the extracted aDNA was genuine, on the basis of mutation rate, but also that the ancient Hissar pea was most likely an early domesticated crop, related to the modern wild pea of a neighbouring region. It is anticipated that this premier extraction of legume aDNA may provide taxonomists with the answers to diverse questions, such as leaf development in legumes, as well as with novel data on the single steps in domesticating legume crops worldwide.

  15. Rapid DNA extraction of pig ear tissues.

    Science.gov (United States)

    Kunhareang, S; Zhou, H; Hickford, J G H

    2010-07-01

    A single-step DNA isolation procedure from pig tissues was developed and the product used directly for polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis and sequencing. The procedure consists of proteinase K digestion of 2-10mg of fresh tissue, at 55 degrees C for 1h, followed by application of the products of digestion to filter paper. A 1.2mm-diameter punch of that paper has sufficient DNA to act as a template for PCR amplification. The quality of the genomic DNA appeared to be high as the PCR amplicons produced sharp banding patterns on both agarose gel electrophoresis and on SSCP analysis, and they could be used for DNA sequencing following cloning. The dried genomic DNA on filter paper can be kept at room temperature. The procedure is considered effective as it is simple, fast and inexpensive. It would be useful for large-scale genotyping and could be used to obtain genomic DNA from various tissues.

  16. Human Papillomavirus (HPV) Vaccines

    Science.gov (United States)

    ... Directory Cancer Prevention Overview Research Human Papillomavirus (HPV) Vaccines On This Page What are human papillomaviruses? Which ... infections? Can HPV infections be prevented? What HPV vaccines are available? Who should get the HPV vaccines? ...

  17. Chromogenic In Situ Hybridization and p16/Ki67 Dual Staining on Formalin-Fixed Paraffin-Embedded Cervical Specimens: Correlation with HPV-DNA Test, E6/E7 mRNA Test, and Potential Clinical Applications

    Directory of Open Access Journals (Sweden)

    Roberta Zappacosta

    2013-01-01

    Full Text Available Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16INK4a/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+. p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16INK4a/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16INK4a/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

  18. Chromogenic in situ hybridization and p16/Ki67 dual staining on formalin-fixed paraffin-embedded cervical specimens: correlation with HPV-DNA test, E6/E7 mRNA test, and potential clinical applications.

    Science.gov (United States)

    Zappacosta, Roberta; Colasante, Antonella; Viola, Patrizia; D'Antuono, Tommaso; Lattanzio, Giuseppe; Capanna, Serena; Gatta, Daniela Maria Pia; Rosini, Sandra

    2013-01-01

    Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16(INK4a)/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16(INK4a)/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16(INK4a)/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

  19. DNA extraction from benthic Cyanobacteria: comparative assessment and optimization.

    Science.gov (United States)

    Gaget, V; Keulen, A; Lau, M; Monis, P; Brookes, J D

    2017-01-01

    Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing. © 2016 The Society for Applied Microbiology.

  20. Residual soil DNA extraction increases the discriminatory power between samples.

    Science.gov (United States)

    Young, Jennifer M; Weyrich, Laura S; Clarke, Laurence J; Cooper, Alan

    2015-06-01

    Forensic soil analysis relies on capturing an accurate and reproducible representation of the diversity from limited quantities of soil; however, inefficient DNA extraction can markedly alter the taxonomic abundance. The performance of a standard commercial DNA extraction kit (MOBIO PowerSoil DNA Isolation kit) and three modified protocols of this kit: soil pellet re-extraction (RE); an additional 24-h lysis incubation step at room temperature (RT); and 24-h lysis incubation step at 55°C (55) were compared using high-throughput sequencing of the internal transcribed spacer I ribosomal DNA. DNA yield was not correlated with fungal diversity and the four DNA extraction methods displayed distinct fungal community profiles for individual samples, with some phyla detected exclusively using the modified methods. Application of a 24 h lysis step will provide a more complete inventory of fungal biodiversity, and re-extraction of the residual soil pellet offers a novel tool for increasing discriminatory power between forensic soil samples.

  1. Evaluation of DNA extraction methods for freshwater eukaryotic microalgae.

    Science.gov (United States)

    Eland, Lucy E; Davenport, Russell; Mota, Cesar R

    2012-10-15

    The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy(®) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended.

  2. Automated genomic DNA extraction from saliva using the QIAxtractor.

    Science.gov (United States)

    Keijzer, Henry; Endenburg, Silvia C; Smits, Marcel G; Koopmann, Miriam

    2010-05-01

    Venipuncture is an invasive procedure to obtain whole blood in order to obtain high quality and sufficient amounts of genomic DNA. Obtaining DNA from non-invasive sources is preferred by patients, medical doctors and researchers. Saliva collected with cotton swabs (Salivette) is increasingly being used to study chemical compounds, and it can also be a source of DNA. However, extracting DNA from Salivettes is very laborious and time consuming. Therefore, we developed a protocol for automated genomic DNA extraction from saliva collected in Salivette using the QIAxtractor. Saliva (0.1-2.0 mL) was collected by chewing on a Salivette for 1-2 min. A total of 70 samples, collected from healthy volunteers, were extracted with the QIAxtractor robot and a Qiagen DX reagent pack. Quantity and quality was assessed using UV spectrometry and real-time polymerase chain reaction (PCR) (substitution at position -729 in the CYP1A2 gene). The average DNA concentration from the saliva samples was 6.0 microg/mL (95% CI 5.4-6.6 microg/mL). In 100% of the saliva samples, PCR products were detected with an average cycle threshold of 23.1 (95% CI 22.6-23.6). DNA can be extracted in sufficient amounts from Salivette with a fully automated system with a short turnaround time. Real-time PCR can be performed with these samples.

  3. DNA extraction protocol for rapid PCR detection of pathogenic bacteria.

    Science.gov (United States)

    Brewster, Jeffrey D; Paoli, George C

    2013-11-01

    Twelve reagents were evaluated to develop a direct DNA extraction method suitable for PCR detection of foodborne bacterial pathogens. Many reagents exhibited strong PCR inhibition, requiring significant dilution of the extract with a corresponding reduction in sensitivity. Most reagents also exhibited much lower recovery of DNA from the gram-positive test organism (Listeria monocytogenes) than from the gram-negative organism (Escherichia coli O157:H7), preventing unbiased detection and quantitation of both organisms. The 5× HotSHOT+Tween reagent exhibited minimal inhibition and high extraction efficiency for both test organisms, providing a 15-min single-tube DNA-extraction protocol suitable for highly sensitive quantitative PCR assays. Published by Elsevier Inc.

  4. High-throughput DNA extraction of forensic adhesive tapes.

    Science.gov (United States)

    Forsberg, Christina; Jansson, Linda; Ansell, Ricky; Hedman, Johannes

    2016-09-01

    Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/μL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  5. Extraction DNA from Activated Sludge-Comparing with Soil Sample

    Institute of Scientific and Technical Information of China (English)

    谢冰; 奚旦立; 陈季华

    2003-01-01

    DNA directly extraction from activated sludge and soil sample with enzyme lyses methods was investigated in this paper. DNA yield from activated sludge was 3.0 mg/g. VLSS, and 28.2-43.8 μg/g soil respectively. The resulting DNA is suitable for PCR.By studied methods, higher quality and quantity of sludge DNA could be obtained rapidly and inexpensively from large number of samples, and the PCR product obtained from this protocol was not affected by contaminated higher concentration of heavy metals.

  6. A LONGITUDINAL STUDY OF HPV16 L1, E6 AND E7 SEROPOSITIVITY AND ORAL HPV16 INFECTION

    Science.gov (United States)

    Beachler, Daniel C.; Viscidi, Raphael; Sugar, Elizabeth A.; Minkoff, Howard; Strickler, Howard D.; Cranston, Ross D.; Wiley, Dorothy J.; Jacobson, Lisa P.; Weber, Kathleen M.; Margolick, Joseph B.; Reddy, Susheel; Gillison, Maura L.; D’Souza, Gypsyamber

    2014-01-01

    Background Individuals with HPV infections can develop IgG antibodies to HPV proteins including the L1 capsid and E6 and E7 oncoproteins. Evidence on whether L1 antibodies reduce the risk of cervical HPV infection is mixed, but this has not been explored for oral HPV infections. Antibodies to HPV16’s E6 oncoprotein have been detected in some oropharyngeal cancer cases years prior to cancer diagnosis, but it is unknown if these antibodies are associated with oral HPV16 DNA. Methods Enzyme linked immunosorbent assays tested for serum antibodies to HPV16’s L1 capsid in 463 HIV-infected and 293 HIV-uninfected adults, and for antibodies to recombinantly expressed E6 and E7 oncoproteins to HPV16 in 195 HIV-infected and 69 HIV-uninfected cancer-free participants at baseline. Oral rinse samples were collected semi-annually for up to three years and tested for HPV DNA using PGMY 09/11 primers. Adjusted Poisson, logistic, and Wei-Lin-Weissfeld regression models were utilized. Results HPV16 L1 seroreactivity did not reduce the subsequent risk of incident oral HPV16 infection in unadjusted (HR=1.4, 95%CI=0.59–3.3) or adjusted (aHR=1.1, 95%CI=0.41–3.0) analysis. Antibodies to HPV16 E6 and E7 oncoproteins were detected in 7.6% and 3.4% of participants respectively, but they were not associated with baseline oral HPV16 DNA prevalence or oral HPV16 persistence (each p-value>0.40). Conclusions Naturally acquired HPV16 L1 antibodies did not reduce the risk of subsequent oral HPV16 infection. HPV16 E6 and E7 seropositivity was not a marker for oral HPV16 infection in this population without HPV-related cancer. PMID:25585068

  7. Acceptability of self-collection sampling for HPV-DNA testing in low-resource settings: a mixed methods approach.

    Science.gov (United States)

    Bansil, Pooja; Wittet, Scott; Lim, Jeanette L; Winkler, Jennifer L; Paul, Proma; Jeronimo, Jose

    2014-06-12

    Vaginal self-sampling with HPV-DNA tests is a promising primary screening method for cervical cancer. However, women's experiences, concerns and the acceptability of such tests in low-resource settings remain unknown. In India, Nicaragua, and Uganda, a mixed-method design was used to collect data from surveys (N = 3,863), qualitative interviews (N = 72; 20 providers and 52 women) and focus groups (N = 30 women) on women's and providers' experiences with self-sampling, women's opinions of sampling at home, and their future needs. Among surveyed women, 90% provided a self- collected sample. Of these, 75% reported it was easy, although 52% were initially concerned about hurting themselves and 24% were worried about not getting a good sample. Most surveyed women preferred self-sampling (78%). However it was not clear if they responded to the privacy of self-sampling or the convenience of avoiding a pelvic examination, or both. In follow-up interviews, most women reported that they didn't mind self-sampling, but many preferred to have a provider collect the vaginal sample. Most women also preferred clinic-based screening (as opposed to home-based self-sampling), because the sample could be collected by a provider, women could receive treatment if needed, and the clinic was sanitary and provided privacy. Self-sampling acceptability was higher when providers prepared women through education, allowed women to examine the collection brush, and were present during the self-collection process. Among survey respondents, aids that would facilitate self-sampling in the future were: staff help (53%), additional images in the illustrated instructions (31%), and a chance to practice beforehand with a doll/model (26%). Self-and vaginal-sampling are widely acceptable among women in low-resource settings. Providers have a unique opportunity to educate and prepare women for self-sampling and be flexible in accommodating women's preference for self-sampling.

  8. Analytical characterization of the APTIMA HPV Assay.

    Science.gov (United States)

    Dockter, Janel; Schroder, Astrid; Eaton, Barbara; Wang, Ann; Sikhamsay, Nathan; Morales, Liezel; Giachetti, Cristina

    2009-07-01

    Human papillomavirus (HPV) testing has improved the sensitivity for the detection of cervical pre-cancer and cancer as compared to Pap testing. Several HPV tests are commercially available and most target the DNA from 13 or 14 high-risk HPV types. The APTIMA HPV Assay however, detects HPV E6/E7 mRNA from 14 high-risk types of HPV: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. To determine the analytical performance characteristics of the APTIMA HPV Assay. Analytical sensitivity, analytical specificity, reproducibility, and the effect of potentially interfering substances was determined for the APTIMA HPV Assay on both the DTS (semi-automated) and TIGRIS DTS (fully automated) systems. The 95% detection limit for both systems was between 17 and 488 copies/reaction, depending on the HPV type. The assay did not cross-react with normal flora and opportunistic organisms that may be found in cervical samples, or low-risk HPV types. Spermicides, anti-fungal and anti-itch medications, whole blood, glacial acetic acid, and most lubricants did not interfere with assay performance. Those lubricants containing polyquaternium 15 did interfere with assay performance. Inter-instrument, inter-operator, inter-lot, and inter-run signal variability were 99% of the data. Intra-run variability was HPV Assay showed excellent performance and robustness.

  9. Highly efficient DNA extraction method from skeletal remains

    Directory of Open Access Journals (Sweden)

    Irena Zupanič Pajnič

    2011-03-01

    Full Text Available Background: This paper precisely describes the method of DNA extraction developed to acquire high quality DNA from the Second World War skeletal remains. The same method is also used for molecular genetic identification of unknown decomposed bodies in routine forensic casework where only bones and teeth are suitable for DNA typing. We analysed 109 bones and two teeth from WWII mass graves in Slovenia. Methods: We cleaned the bones and teeth, removed surface contaminants and ground the bones into powder, using liquid nitrogen . Prior to isolating the DNA in parallel using the BioRobot EZ1 (Qiagen, the powder was decalcified for three days. The nuclear DNA of the samples were quantified by real-time PCR method. We acquired autosomal genetic profiles and Y-chromosome haplotypes of the bones and teeth with PCR amplification of microsatellites, and mtDNA haplotypes 99. For the purpose of traceability in the event of contamination, we prepared elimination data bases including genetic profiles of the nuclear and mtDNA of all persons who have been in touch with the skeletal remains in any way. Results: We extracted up to 55 ng DNA/g of the teeth, up to 100 ng DNA/g of the femurs, up to 30 ng DNA/g of the tibias and up to 0.5 ng DNA/g of the humerus. The typing of autosomal and YSTR loci was successful in all of the teeth, in 98 % dekalof the femurs, and in 75 % to 81 % of the tibias and humerus. The typing of mtDNA was successful in all of the teeth, and in 96 % to 98 % of the bones. Conclusions: We managed to obtain nuclear DNA for successful STR typing from skeletal remains that were over 60 years old . The method of DNA extraction described here has proved to be highly efficient. We obtained 0.8 to 100 ng DNA/g of teeth or bones and complete genetic profiles of autosomal DNA, Y-STR haplotypes, and mtDNA haplotypes from only 0.5g bone and teeth samples.

  10. Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize.

    Science.gov (United States)

    Abdel-Latif, Amani; Osman, Gamal

    2017-01-01

    The world's top three cereals, based on their monetary value, are rice, wheat, and corn. In cereal crops, DNA extraction is difficult owing to rigid non-cellulose components in the cell wall of leaves and high starch and protein content in grains. The advanced techniques in molecular biology require pure and quick extraction of DNA. The majority of existing DNA extraction methods rely on long incubation and multiple precipitations or commercially available kits to produce contaminant-free high molecular weight DNA. In this study, we compared three different methods used for the isolation of high-quality genomic DNA from the grains of cereal crop, Zea mays, with minor modifications. The DNA from the grains of two maize hybrids, M10 and M321, was extracted using extraction methods DNeasy Qiagen Plant Mini Kit, CTAB-method (with/without 1% PVP) and modified Mericon extraction. Genes coding for 45S ribosomal RNA are organized in tandem arrays of up to several thousand copies and contain codes for 18S, 5.8S and 26S rRNA units separated by internal transcribed spacers ITS1 and ITS2. While the rRNA units are evolutionary conserved, ITS regions show high level of interspecific divergence and have been used frequently in genetic diversity and phylogenetic studies. In this study, the genomic DNA was then amplified with PCR using primers specific for ITS gene. PCR products were then visualized on agarose gel. The modified Mericon extraction method was found to be the most efficient DNA extraction method, capable to provide high DNA yields with better quality, affordable cost and less time.

  11. Modification of gelatin-DNA interaction for optimised DNA extraction from gelatin and gelatin capsule.

    Science.gov (United States)

    Mohamad, Nurhidayatul Asma; Mustafa, Shuhaimi; El Sheikha, Aly Farag; Khairil Mokhtar, Nur Fadhilah; Ismail, Amin; Ali, Md Eaqub

    2016-05-01

    Poor quality and quantity of DNA extracted from gelatin and gelatin capsules often causes failure in the determination of animal species using PCR. Gelatin, which is mainly derived from porcine and bovine, has been a matter of concern among customers in order to fulfill religious obligation and safety precaution against several transmissible infectious diseases associated with bovine species. Thus, optimised DNA extraction from gelatin is very important for successful real-time PCR detection of gelatin species. In this work, the DNA extraction method was optimised in terms of lysis incubation period and inclusion of pre-treatment pH modification of samples. The yield of DNA extracted from porcine gelatin was significantly increased when the pH of the samples was adjusted to pH 8.5 prior to DNA precipitation with isopropanol. The optimal pH for DNA precipitation from bovine gelatin solution was then determined at the original pH range of solution: pH 7.6 to 8. A DNA fragment of approximately 300 base pairs was available for PCR amplification. DNA extracted from gelatin and commercially available capsules has been successfully utilised for species detection using real-time PCR assay. However, significant adulterations of porcine and bovine in pure gelatin and capsules have been detected, which require further analytical techniques for validation. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  12. Acceptability of self-collected versus provider-collected sampling for HPV DNA testing among women in rural El Salvador.

    Science.gov (United States)

    Rosenbaum, Alan J; Gage, Julia C; Alfaro, Karla M; Ditzian, Lauren R; Maza, Mauricio; Scarinci, Isabel C; Felix, Juan C; Castle, Philip E; Villalta, Sofia; Miranda, Esmeralda; Cremer, Miriam L

    2014-08-01

    To determine the acceptability of self-collected versus provider-collected sampling among women participating in public sector HPV-based cervical cancer screening in El Salvador. Two thousand women aged 30-49 years underwent self-collected and provider-collected sampling with careHPV between October 2012 and March 2013 (Qiagen, Gaithersburg, MD, USA). After sample collection, a random sample of women (n=518) were asked about their experience. Participants were questioned regarding sampling method preference, previous cervical cancer screening, HPV and cervical cancer knowledge, HPV risk factors, and demographic information. All 518 women approached to participate in this questionnaire study agreed and were enrolled, 27.8% (142 of 511 responding) of whom had not received cervical cancer screening within the past 3 years and were considered under-screened. Overall, 38.8% (n=201) preferred self-collection and 31.9% (n=165) preferred provider collection. Self-collection preference was associated with prior tubal ligation, HPV knowledge, future self-sampling preference, and future home-screening preference (P<0.05). Reasons for self-collection preference included privacy/embarrassment, ease, and less pain; reasons cited for provider-collection preference were result accuracy and provider knowledge/experience. Self-sampling was found to be acceptable, therefore screening programs could consider offering this option either in the clinic or at home. Self-sampling at home may increase coverage in low-resource countries and reduce the burden that screening places upon clinical infrastructure. Copyright © 2014 International Federation of Gynecology and Obstetrics. All rights reserved.

  13. Overcoming DNA extraction problems from carnivorous plants

    OpenAIRE

    Fleischmann, Andreas; Heubl, Günther

    2009-01-01

    We tested previously published protocols for DNA isolation from plants with high contents of polyphenols and polysaccharides for several taxa of carnivorous plants. However, we did not get satisfying results with fresh or silica dried leaf tissue obtained from field collected or greenhouse grown plants, nor from herbarium specimens. Therefore, we have developed a simple modified protocol of the commercially available Macherey- Nagel NucleoSpin® Plant kit for rapid, effective and reproducible ...

  14. Human papilloma viruses (HPV and breast cancer.

    Directory of Open Access Journals (Sweden)

    James Sutherland Lawson

    2015-12-01

    Full Text Available Purpose: Human papillomaviruses (HPV may have a role in some breast cancers. The purpose of this study is to fill important gaps in the evidence. These gaps are: (i confirmation of the presence of high risk for cancer HPVs in breast cancers, (ii evidence of HPV infections in benign breast tissues prior to the development of HPV positive breast cancer in the same patients, (iii evidence that HPVs are biologically active and not harmless passengers in breast cancer.Methods: RNA-seq data from The Cancer Genome Atlas (TCGA was used to identify HPV RNA sequences in breast cancers. We also conducted a retrospective cohort study based on polymerase chain reaction (PCR analyses to identify HPVs in archival specimens from Australian women with benign breast biopsies who later developed breast cancer. To assess whether HPVs in breast cancer were biologically active, the expression of the oncogenic protein HPV E7 was assessed by immunohistochemistry (IHC.Results: Thirty (3.5% low risk and 20 (2.3% high risk HPV types were identified in 855 breast cancers from the TCGA data base. The high risk types were HPV 18 (48%, HPV 113 (24%, HPV 16 (10%, HPV 52 (10%. Data from the PCR cohort study, indicated that HPV type 18 was the most common type identified in breast cancer specimens (55% of 40 breast cancer specimens followed by HPV 16 (13%. The same HPV type was identified in both the benign and subsequent breast cancer in 15 patients. HPV E7 proteins were identified in 72% of benign breast specimens and 59% of invasive breast cancer specimens.Conclusions: There were 4 observations of particular interest: (i confirmation by both NGS and PCR of the presence of high risk HPV gene sequences in breast cancers, (ii a correlation between high risk HPV in benign breast specimens and subsequent HPV positive breast cancer in the same patient, (iii HPVs in breast cancer are likely to be biologically active (as shown by transcription of HPV DNA to RNA plus the expression of

  15. Automated DNA extraction of single dog hairs without roots for mitochondrial DNA analysis.

    Science.gov (United States)

    Bekaert, Bram; Larmuseau, Maarten H D; Vanhove, Maarten P M; Opdekamp, Anouschka; Decorte, Ronny

    2012-03-01

    Dogs are intensely integrated in human social life and their shed hairs can play a major role in forensic investigations. The overall aim of this study was to validate a semi-automated extraction method for mitochondrial DNA analysis of telogenic dog hairs. Extracted DNA was amplified with a 95% success rate from 43 samples using two new experimental designs in which the mitochondrial control region was amplified as a single large (± 1260 bp) amplicon or as two individual amplicons (HV1 and HV2; ± 650 and 350 bp) with tailed-primers. The results prove that the extraction of dog hair mitochondrial DNA can easily be automated to provide sufficient DNA yield for the amplification of a forensically useful long mitochondrial DNA fragment or alternatively two short fragments with minimal loss of sequence in case of degraded samples.

  16. Proboscidean DNA from museum and fossil specimens: an assessment of ancient DNA extraction and amplification techniques.

    Science.gov (United States)

    Yang, H; Golenberg, E M; Shoshani, J

    1997-06-01

    Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.

  17. Nonhomologous DNA End Joining in Cell-Free Extracts

    OpenAIRE

    Sheetal Sharma; Raghavan, Sathees C.

    2010-01-01

    Among various DNA damages, double-strand breaks (DSBs) are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  18. Nonhomologous DNA End Joining in Cell-Free Extracts

    Directory of Open Access Journals (Sweden)

    Sheetal Sharma

    2010-01-01

    Full Text Available Among various DNA damages, double-strand breaks (DSBs are considered as most deleterious, as they may lead to chromosomal rearrangements and cancer when unrepaired. Nonhomologous DNA end joining (NHEJ is one of the major DSB repair pathways in higher organisms. A large number of studies on NHEJ are based on in vitro systems using cell-free extracts. In this paper, we summarize the studies on NHEJ performed by various groups in different cell-free repair systems.

  19. DNA Extraction from Eriocaulon Plants and Construction of RAPD System

    Institute of Scientific and Technical Information of China (English)

    Xue Xian; Lin Shanzhi; Zhang Zhixiang

    2004-01-01

    There have been many arguments on the classification of Eriocaulon Linn. by morphology so far, and little is known about the use of molecular marker for genetic for genetic diversity of Eriocaulon plants. To apply the technique of molecular marker to the research of genetic diversity of Eriocaulon plants, the study of the extraction method of DNA from the Eriocaulon plants and the RAPD system are essential for researchers. In this paper, the extraction of genome DNA from the silica-gel-dried leaves of several species of Eriocaulon distributed in China was studied, and the best RAPD analysis technique condition of Eriocaulon plants was analyzed.

  20. An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts.

    Science.gov (United States)

    Kallmeyer, Jens; Smith, David C

    2009-07-01

    We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.

  1. Prognostic evaluation of DNA index in HIV-HPV co-infected women cervical samples attending in reference centers for HIV-AIDS in Recife.

    Directory of Open Access Journals (Sweden)

    Albert Eduardo Silva Martins

    Full Text Available INTRODUCTION: Persistence of cervical infection caused by human papillomavirus (HPV types with high oncogenic risk may lead to cervical intraepithelial neoplasia (CIN. The aim of the present study was to evaluate whether, in HIV-positive women, the presence of aneuploidy in cervical cell samples is associated with presence and evolution of CIN. METHODS: The present study had two stages. In the first stage, comprising a cross-sectional study, the association between the presence of aneuploidy seen via flow cytometry and sociodemographic characteristics, habits and characteristics relating to HPV and HIV infection was analyzed. In the second stage, comprising a cohort study, it was investigated whether aneuploidy was predictive of CIN evolution. RESULTS: No association was observed between the presence of aneuploidy and HPV infection, or between its presence and alterations seen in oncotic cytological analysis. On the other hand, aneuploidy was associated with the presence of CIN (p = 0.030 in histological analysis and with nonuse of antiretroviral therapy (p = 0.001. Most of the HIV-positive women (234/272 presented normal CD4+ T lymphocyte counts (greater than 350 cells/mm3 and showed a greater aneuploidy regression rate (77.5% than a progression rate (23.9% over a follow-up of up to two years. CONCLUSION: Although there was an association between the presence of cervical tissue lesions and the DNA index, the latter was not predictive of progression of the cervical lesion. This suggests that progression of the cervical lesion to cancer in HIV-positive women may also be changed through improvement of the immunological state enabled by using antiretroviral therapy.

  2. Plant and metagenomic DNA extraction of mucilaginous seeds.

    Science.gov (United States)

    Ramos, Simone N M; Salazar, Marcela M; Pereira, Gonçalo A G; Efraim, Priscilla

    2014-01-01

    The pulp surrounding the seeds of some fruits is rich in mucilage, carbohydrates, etc. Some seeds are rich in proteins and polyphenols. Fruit seeds, like cacao (Theobroma cacao) and cupuassu (Theobroma grandiflorum), are subjected to fermentation to develop flavor. During fermentation, ethanol is produced [2-6]. All of these compounds are considered as interfering substances that hinder the DNA extraction [4-8]. Protocols commonly used in the DNA extraction in samples of plant origin were used, but without success. Thus, a protocol for DNA samples under different conditions that can be used for similar samples was developed and applied with success. The protocol initially described for RNA samples by Zeng et al. [9] and with changes proposed by Provost et al. [5] was adapted for extracting DNA samples from those described. However, several modifications have been proposed:•Samples were initially washed with petroleum ether for fat phase removal.•RNAse was added to the extraction buffer, while spermidin was removed.•Additional steps of extraction with 5 M NaCl, saturated NaCl and CTAB (10%) were included and precipitation was carried out with isopropanol, followed by washing with ethanol.

  3. Comparison of DNA and RNA extraction methods for mummified tissues.

    Science.gov (United States)

    Konomi, Nami; Lebwohl, Eve; Zhang, David

    2002-12-01

    Nucleic acids extracted from mummified tissues are valuable materials for the study of ancient human beings. Significant difficulty in extracting nucleic acids from mummified tissues has been reported due to chemical modification and degradation. The goal of this study was to determine a method that is more efficient for DNA and RNA extraction from mummified tissues. Twelve mummy specimens were analyzed with 9 different nucleic acid extraction methods, including guanidium thiocyanate (GTC) and proteinase K/detergent based methods prepared in our laboratory or purchased. Glyceraldehyde 3-phosphate dehydrogenase DNA and beta-actin RNA were used as markers for the presence of adequate DNA and RNA, respectively, for PCR and RT-PCR amplification. Our results show that 5 M GTC is more efficient of releasing nucleic acids from mummified tissue than proteinase K/detergent, and phenol/chloroform extraction with an additional chloroform step is more efficient than phenol/chloroform along. We were able to isolate DNAs from all 12 specimens and RNAs from 8 of 12 specimens, and the nucleic acids were sufficient for PCR and RT-PCR analysis. We further tested hepatitis viruses including hepatitis B virus, hepatitis C virus, hepatitis G virus, and TT virus DNA, and fail to detect these viruses in all 12 specimens.

  4. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura

    2013-01-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  5. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura;

    2013-01-01

    protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA......Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction...... from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore...

  6. Direct RAPD evaluation of bacteria without conventional DNA extraction

    Directory of Open Access Journals (Sweden)

    Welington Luiz Araújo

    2004-07-01

    Full Text Available The present work reports successful DNA amplification of Pantoea agglomerans and Bacillus pumilus through Random Amplified Polymorphic DNA (RAPD. For this, template DNA was obtained without conventional DNA extraction. The procedure was as follows: cultures grown for 20 hours in 5 mL LB medium were centrifuged and the resulting preparation was suspended in TE buffer. After boiling, the cell suspension was diluted and 2.0 µl were used in reactions of 15 µl. The results showed no significant differences among the RAPD profile of the PCR reactions derived from the boiling and phenol extraction methods, suggesting the utilization of this method for genetic population analysis.O presente trabalho mostra a amplificação de DNA das bactérias Pantoea agglomerans e Bacillus pumilus por meio da técnica de RAPD (Amplificação ao acaso de DNA polimórfico. Para esta análise, o DNA molde foi obtido sem a utilização de técnicas de extração convencional, ou seja, sem a purificação do DNA. Bactérias foram cultivadas por 20 horas em 5 mL de meio LB, centrifugado e ressuspendido em tampão TE. A suspensão resultante foi fervida por 5 min., diluída e 2,0 µL foram usados em reações de 15 µL. Os resultados mostraram que os padrões observados com o DNA obtido pela fervura das células não apresentou diferenças significativas daquele obtido com DNA extraído e purificado com fenol, sugerindo a possibilidade da utilização deste método para o estudo da variabilidade genética de populações microbianas.

  7. AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples.

    Science.gov (United States)

    Liu, Jason Y; Zhong, Chang; Holt, Allison; Lagace, Robert; Harrold, Michael; Dixon, Alan B; Brevnov, Maxim G; Shewale, Jaiprakash G; Hennessy, Lori K

    2012-07-01

    The AutoMate Express™ Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler™ lysis buffer or PrepFiler BTA™ lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep™ column, the lysate in the sample tubes were loaded onto AutoMate Express™ instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework-type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day-to-day operation. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

  8. HPV Associated Head and Neck Cancer.

    Science.gov (United States)

    Spence, Tara; Bruce, Jeff; Yip, Kenneth W; Liu, Fei-Fei

    2016-08-05

    Head and neck cancers (HNCs) are a highly heterogeneous group of tumours that are associated with diverse clinical outcomes. Recent evidence has demonstrated that human papillomavirus (HPV) is involved in up to 25% of HNCs; particularly in the oropharyngeal carcinoma (OPC) subtype where it can account for up to 60% of such cases. HPVs are double-stranded DNA viruses that infect epithelial cells; numerous HPV subtypes, including 16, 18, 31, 33, and 35, drive epithelial cell transformation and tumourigenesis. HPV positive (HPV+) HNC represents a distinct molecular and clinical entity from HPV negative (HPV-) disease; the biological basis for which remains to be fully elucidated. HPV positivity is strongly correlated with a significantly superior outcome; indicating that such tumours should have a distinct management approach. This review focuses on the recent scientific and clinical investigation of HPV+ HNC. In particular, we discuss the importance of molecular and clinical evidence for defining the role of HPV in HNC, and the clinical impact of HPV status as a biomarker for HNC.

  9. Rapid DNA extraction methods and new primers for randomly amplified polymorphic DNA analysis of Giardia duodenalis.

    Science.gov (United States)

    Deng, M Q; Cliver, D O

    1999-08-01

    A randomly amplified polymorphic DNA (RAPD) procedure using simple genomic DNA preparation methods and newly designed primers was optimized for analyzing Giardia duodenalis strains. Genomic DNA was extracted from in vitro cultivated trophozoites by five freezing-thawing cycles or by sonic treatment. Compared to a conventional method involving proteinase K digestion and phenol extraction, both freezing-thawing and sonication were equally efficient, yet with the advantage of being much less time- and labor-intensive. Five of the 10 tested RAPD primers produced reproducible polymorphisms among five human origin G. duodenalis strains, and grouping of these strains based on RAPD profiles was in agreement among these primers. The consistent classification of two standard laboratory reference strains, Portland-1 and WB, in the same group confirmed previous results using other fingerprinting methods, indicating that the reported simple DNA extraction methods and the selected primers are useful in RAPD for molecular characterization of G. duodenalis strains.

  10. Comparison of different methodologies for DNA extraction from Aegla longirostri

    Directory of Open Access Journals (Sweden)

    João Vitor Trindade Bitencourt

    2007-11-01

    Full Text Available The aim of this study was to compare some DNA extraction methodologies for Aegla longirostri. The protocols were based on the traditional phenol-chloroform DNA extraction methodology and using a commercial kit for DNA extraction. They differed in tissues used, the addition - or not - of beta-mercaptoethanol to the lysis buffer, times and methods for the animal's conservation (frozen, in ethanol or fresh. Individuals stored at -20°C for a long time supplied lower molecular weight DNA than those stored for a short time. The best yield for the specimens preserved in ethanol was obtained for 15 days storage in 95% ethanol. The kit resulted in a low quantity of high molecular weight DNA. The best protocol for DNA extraction from Aeglidae, and probably for other crustaceans should, therefore, utilize fresh specimens, with addition of beta-mercaptoethanol to the lysis buffer.Marcadores moleculares são ferramentas úteis para esclarecer dúvidas a respeito dos Aeglidae, único grupo de crustáceos Anomura de água doce. Essas técnicas dependem da obtenção de DNA de boa qualidade e sem contaminantes. O objetivo deste estudo foi comparar algumas metodologias de extração de DNA de Aegla longirostri. Quatorze protocolos foram analisados, baseados na metodologia tradicional de extração de DNA com fenol-clorofórmio, exceto o protocolo K no qual se utilizou um Kit. Os procedimentos diferiram quanto aos tecidos utilizados e a adição de beta-mercaptoetanol ao tampão de lise. Avaliaram-se também diferentes tempos e maneiras de conservação. Indivíduos congelados apresentaram maior degradação do material obtido conforme o tempo em que ficaram congelados. Para os indivíduos conservados em álcool, aqueles mantidos em etanol 95% forneceram material de melhor qualidade. A utilização do Mini Kit resultou em uma quantidade muito pequena de DNA de alto peso molecular. O melhor protocolo para extração de DNA de Aeglidae utilizou músculos e br

  11. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    Directory of Open Access Journals (Sweden)

    Anja Gulliksen

    2012-01-01

    Full Text Available The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28 from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

  12. HPV genotypes concordance between sex partners.

    Science.gov (United States)

    Benevolo, M; Mottolese, M; Marandino, F; Carosi, M; Diodoro, M G; Sentinelli, S; Visca, P; Rollo, F; Mariani, L; Vocaturo, G; Sindico, R; Di Giannuario, D; Perrone Donnorso, R; Pellicciotta, M; Vocaturo, A

    2007-12-01

    The HPV genotype concordance in the sexual couples could support the sexual viral transmission of HPV infection. The present study contains a case-report of a stable Italian sex couple harbouring the same five HPV genotypes in their genital samples. The female partner, affected by vulvar condilomatosis, evidenced positivity in her cervicovaginal scraping with high risk HPV DNA Hybrid Capture 2 test and was negative at liquid-based performed Pap Test and at colposcopic examination. The male partner was clinically healthy regarding his external genitalia. In both male and female genital scrapings, the following HPV genotypes were detected by means of a PCR-based assay: 6, 16, 53, 73 and 84. This considerably high genotype concordance does not appear to be casual and supports, in our opinion, the hypothesis that genital HPV types are sexually transmitted agents

  13. A PCR amplification method without DNA extraction.

    Science.gov (United States)

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  14. A method suitable for DNA extraction from humus-rich soil.

    Science.gov (United States)

    Miao, Tianjin; Gao, Song; Jiang, Shengwei; Kan, Guoshi; Liu, Pengju; Wu, Xianming; An, Yingfeng; Yao, Shuo

    2014-11-01

    A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

  15. Direct Extraction and Amplification of DNA from Soil.

    Science.gov (United States)

    Trevors, Jack T.; Leung, K.

    1998-01-01

    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  16. Direct Extraction and Amplification of DNA from Soil.

    Science.gov (United States)

    Trevors, Jack T.; Leung, K.

    1998-01-01

    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  17. DNA extraction and barcode identification of development stages of forensically important flies in the Czech Republic.

    Science.gov (United States)

    Olekšáková, Tereza; Žurovcová, Martina; Klimešová, Vanda; Barták, Miroslav; Šuláková, Hana

    2017-03-21

    Several methods of DNA extraction, coupled with 'DNA barcoding' species identification, were compared using specimens from early developmental stages of forensically important flies from the Calliphoridae and Sarcophagidae families. DNA was extracted at three immature stages - eggs, the first instar larvae, and empty pupal cases (puparia) - using four different extraction methods, namely, one simple 'homemade' extraction buffer protocol and three commercial kits. The extraction conditions, including the amount of proteinase K and incubation times, were optimized. The simple extraction buffer method was successful for half of the eggs and for the first instar larval samples. The DNA Lego Kit and DEP-25 DNA Extraction Kit were useful for DNA extractions from the first instar larvae samples, and the DNA Lego Kit was also successful regarding the extraction from eggs. The QIAamp DNA mini kit was the most effective; the extraction was successful with regard to all sample types - eggs, larvae, and pupari.

  18. Determination of HPV DNA viral load by hybrid capture assay and its association with cytological findings Determinação da carga viral de DNA de HPV pelo ensaio de captura híbrida e sua associação com achados citológicos

    Directory of Open Access Journals (Sweden)

    Inês Aparecida Tozetti

    2006-12-01

    Full Text Available OBJECTIVE: To compare the relation between HPV viral load by hybrid capture II test (HCII and cytological findings. METHODS: Three hundred sixty-two reagent samples to HPV DNA by HCII had their viral loads classified in four categories and correlated to cytological results. RESULTS: Twenty-two samples (6.1% were reagent only to low-risk oncogenic types (group A and 340 (93.9% were reagent to high-risk oncogenic types (group B. The correlation between viral load for the reagent samples to group A and cytological results showed low-grade squamous intraepithelial lesion (LSIL predominance (50%. Most of this group samples had viral load between 1 to OBJETIVO: Comparar a relação entre a carga viral do HPV por captura híbrida II (HCII e os achados citológicos. METODOS: Trezentas e sessenta e duas amostras reagentes para DNA de HPV por HCII tiveram suas cargas virais classificadas em quatro categorias e correlacionadas aos resultados citológicos. RESULTADOS: Vinte e duas amostras (6,1% foram reagentes somente para os tipos de baixo risco oncogênico (grupo A e 340 (93,9% foram reagentes para os tipos de alto risco oncogênico (grupo B. A correlação entre carga viral das amostras reagentes para o grupo A e resultados citológicos mostrou predominância (50% de lesão escamosa intraepitelial de baixo grau (LSIL. A maioria das amostras desse grupo teve carga viral entre 1 e < 10RLU/PCA. Nos pacientes reagentes para o grupo B observamos que 52,1% tiveram citologia LSIL e 38,2% tiveram citologia negativa para lesão intraepitelial e malignidade (NILM. Os pacientes com LSIL tiveram a carga viral bem distribuída, com ligeira predominância da categoria de 100 a < 1.000RLU/PCB. As amostras com carga viral entre 1 e < 10RLU/PCB mostraram predominância de citologia NILM (48.1%. Lesões escamosas de alto grau (3,4% foram presentes nas amostras com carga viral entre 100 e < 1.000RLU/PCB (p = 0,023. Houve correlação entre a mediana da carga viral para o

  19. Comparison of DNA extraction methods for meat analysis.

    Science.gov (United States)

    Yalçınkaya, Burhanettin; Yumbul, Eylem; Mozioğlu, Erkan; Akgoz, Muslum

    2017-04-15

    Preventing adulteration of meat and meat products with less desirable or objectionable meat species is important not only for economical, religious and health reasons, but also, it is important for fair trade practices, therefore, several methods for identification of meat and meat products have been developed. In the present study, ten different DNA extraction methods, including Tris-EDTA Method, a modified Cetyltrimethylammonium Bromide (CTAB) Method, Alkaline Method, Urea Method, Salt Method, Guanidinium Isothiocyanate (GuSCN) Method, Wizard Method, Qiagen Method, Zymogen Method and Genespin Method were examined to determine their relative effectiveness for extracting DNA from meat samples. The results show that the salt method is easy to perform, inexpensive and environmentally friendly. Additionally, it has the highest yield among all the isolation methods tested. We suggest this method as an alternative method for DNA isolation from meat and meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. DNA vaccine encoding HPV-16 E7 with mutation in L-Y-C-Y-E pRb-binding motif induces potent anti-tumor responses in mice.

    Science.gov (United States)

    Bahrami, Armina Alagheband; Ghaemi, Amir; Tabarraei, Alijan; Sajadian, Azadeh; Gorji, Ali; Soleimanjahi, Hoorieh

    2014-09-01

    Cervical cancer is the second most common cancer among women worldwide and remains a clinical problem despite improvements in early detection and therapy. The human papillomavirus (HPV) type 16 (HPV16) E7 oncoprotein expressed in cervical carcinoma cells are considered as attractive tumor-specific antigen targets for immunotherapy. Since the transformation potential of the oncogenes, vaccination based of these oncogenes is not safe. In present study, DNA vaccine expressing the modified variant with mutation in pRb-binding motif of the HPV-16 E7 oncoprotein was generated. A novel modified E7 gene with mutation in LYCYE motif was designed and constructed and the immunogenicity and antitumor effect of therapeutic DNA vaccines encoding the mutant and wild type of E7 gene were investigated. The L-Y-C-Y-E pRb-binding motif of E7 proteins has been involved in the immortalization and transformation of the host cell. The results showed that the mutant and wild type HPV-16 E7 vectors expressed the desired protein. Furthermore, the immunological mechanism behind mutant E7 DNA vaccine can be attributed at least partially to increased cytotoxic T lymphocyte, accompanied by the up-regulation of Th1-cytokine IFN-γ and TNF-β and down-regulation of Th3-cytokine TGF-β. Immunized mice with mutant plasmid demonstrated significantly stronger cell immune responses and higher levels of tumor protection than wild-type E7 DNA vaccine. The results exhibit that modified E7 DNA vaccine may be a promising candidate for development of therapeutic vaccine against HPV-16 cancers.

  1. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  2. Evaluation of DNA extraction methods of rumen microbial populations.

    Science.gov (United States)

    Villegas-Rivera, Gabriela; Vargas-Cabrera, Yevani; González-Silva, Napoleón; Aguilera-García, Florentino; Gutiérrez-Vázquez, Ernestina; Bravo-Patiño, Alejandro; Cajero-Juárez, Marcos; Baizabal-Aguirre, Víctor Manuel; Valdez-Alarcón, Juan José

    2013-02-01

    The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.

  3. Papanicolau smear chances to be diagnostic for cervical squamous intraepithelial lesions (SIL) with or without detectable HPV DNA at in situ hybridization analysis.

    Science.gov (United States)

    Sopracordevole, F; Cadorin, L; Muffato, G; De Benetti, L; Parin, A

    1993-01-01

    The Authors have correlated 39 cervical diagnostic biopsies for squamous intraepithelial lesions (SILs) with correspective Papanicolau smears (PS), with relation to the presence or the absence of HPV of oncogenic type (HPV-one) detected by in situ hybridization (ISH). Agreement between cytological and histological diagnosis was present in 14 of 16 cases with detectable HPV-one and only in 12 of 23 cases without detectable HPV-one at ISH. The importance of the HPV type in the SILs with relation to the diagnostic accuracy of Papanicolaou smears has been discussed.

  4. Human Papillomavirus (HPV)

    Science.gov (United States)

    ... Español Text Size Email Print Share Human Papillomavirus (HPV) Page Content Article Body According to the Centers ... and how to prevent it. How to Prevent HPV: There are 3 types of HPV vaccine: Cervarix , ...

  5. PCR-based typing of DNA extracted from cigarette butts.

    Science.gov (United States)

    Hochmeister, M N; Budowle, B; Jung, J; Borer, U V; Comey, C T; Dirnhofer, R

    1991-01-01

    Limited genetic marker information can be obtained from saliva by typing by conventional serological means. Thus, the application of PCR-based DNA typing methods was investigated as a potential approach for typing genetic markers in saliva. DNA was isolated from 200 cigarettes smoked by 10 different individuals (20 cigarettes per individual) and from 3 cigarette butts recovered from 2 crime scenes (adjudicated cases) using a Chelex 100 extraction procedure. The amount of recovered human DNA was quantified by slot-blot analysis and ranged from approximately less than 2-160 ng DNA per cigarette butt for the 200 samples, and 8 ng, 50 ng, and 100 ng for the cigarette butts from the adjudicated cases. The DNA was successfully amplified by the polymerase chain reaction (PCR) for the HLA-DQ alpha locus (99 out of 100 samples) as well as for the variable number of tandem repeat (VNTR) locus D1S80 (99 out of 100 samples). Amplification and typing of DNA was successful on all samples recovered from the crime scenes. The results suggest that PCR-based typing of DNA offers a potential method for genetically characterizing traces of saliva on cigarette butts.

  6. Rapid methods to extract DNA and RNA from Cryptococcus neoformans.

    Science.gov (United States)

    Bolano, A; Stinchi, S; Preziosi, R; Bistoni, F; Allegrucci, M; Baldelli, F; Martini, A; Cardinali, G

    2001-12-01

    Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.

  7. Chlamydia trachomatis prevalence and chlamydial/HPV co-infection among HPV-unvaccinated young Italian females with normal cytology.

    Science.gov (United States)

    Panatto, Donatella; Amicizia, Daniela; Bianchi, Silvia; Frati, Elena Rosanna; Zotti, Carla Maria; Lai, Piero Luigi; Domnich, Alexander; Colzani, Daniela; Gasparini, Roberto; Tanzi, Elisabetta

    2015-01-01

    Infections caused by Chlamydia trachomatis (Ct) and human papillomavirus (HPV) are the two main sexually transmitted infections; however, epidemiological data on Ct prevalence and Ct/HPV co-infection in Italy are scant. This study aimed at estimating the prevalence of Ct infection and Ct/HPV co-infection in young HPV-unvaccinated females with normal cytology, and placed particular attention on the possible association between Ct-DNA positivity and different HPV infecting genotypes. Five hundred 66 healthy females aged 16-26 years without cervical lesions, previously assessed for HPV infection (HPV-DNA prevalence: 18.2%), were tested for Ct-DNA. The overall prevalence of Ct was 5.8% (95% CI: 4.2-8.1), while Ct/HPV co-infection was recorded in 2.7% (95% CI: 1.6-4.3) of subjects. Compared with HPV-DNA-negative females, HPV-DNA positive subjects had significantly (P < 0.001) higher odds of being infected with Ct (odds ratio of 4.20, 95% CI: 2.01-8.71). Both Ct and Ct/HPV infections were much more prevalent in under 18-year-olds than in older women. Subjects positive for single high-risk HPV genotypes and various multiple HPV infections had higher odds of being Ct-DNA positive. Our findings confirm that HPV and Ct infections are very common among asymptomatic young Italian females. This underlines the urgent need for nationwide Ct screening programs and reinforcement of sexual health education, which would be the most important public health strategies, since no Ct vaccines are currently available.

  8. DNA Everywhere. A Guide for Simplified Environmental Genomic DNA Extraction Suitable for Use in Remote Areas

    Energy Technology Data Exchange (ETDEWEB)

    Pecora, Gabrielle N. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Reid, Francine C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Tom, Lauren M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Piceno, Yvette M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Andersen, Gary L. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2016-05-01

    Collecting field samples from remote or geographically distant areas can be a financially and logistically challenging. With participation of a local organization where the samples are originated from, gDNA samples can be extracted from the field and shipped to a research institution for further processing and analysis. The ability to set up gDNA extraction capabilities in the field can drastically reduce cost and time when running long-term microbial studies with a large sample set. The method outlined here has developed a compact and affordable method for setting up a “laboratory” and extracting and shipping gDNA samples from anywhere in the world. This white paper explains the process of setting up the “laboratory”, choosing and training individuals with no prior scientific experience how to perform gDNA extractions and safe methods for shipping extracts to any research institution. All methods have been validated by the Andersen group at Lawrence Berkeley National Laboratory using the Berkeley Lab PhyloChip.

  9. Human papilloma virus (HPV genotypes prevalence in a region of South Italy (Apulia

    Directory of Open Access Journals (Sweden)

    Maria Franca Coscia

    2015-09-01

    Full Text Available INTRODUCTION. Since human papillomavirus (HPV is the central casual factor in cervical cancer, understanding the epidemiology and geographical area distribution of the most prevalent HPV genotypes constitutes an important step towards development of strategies of prevention. AIM. The aim of this study was to investigate the prevalence of HPV infection and to determine HPV types distribution among 822 HPV positive women and some sexual male partners in Apulia (Italy. METHODS. HPV DNA detection and genotyping was performed by nested-PCR for the L1 region and reverse line blot hybridization allowing the specific detection of 24 HPV genotyping both high risk (HR and low risk (LR. RESULTS. The most prevalent HPV genotypes were HPV 16 (35%, HPV 31 (16% HPV 6 (9%, HPV 58 and 66 (7%, followed by HPV 33 (6%, HPV 18 and 56 (4%, HPV 70 and 45 (3%, HPV 53 and 11 (2%. Currently 1.5% of tested specimens remained unclassified. Multiple infections with at last two different high-risk HPV genotypes were observed in 10% of specimens. CONCLUSIONS. This finding adds knowledge to HPV epidemiological investigation, and addresses further studies aimed to consider public health for identifying groups at risk for cervical cancer.

  10. Comparison of eight methods of genomic DNA extraction from babassu.

    Science.gov (United States)

    Viana, J P G; Borges, A N C; Lopes, A C A; Gomes, R L F; Britto, F B; Lima, P S C; Valente, S E S

    2015-12-22

    Babassu (Orbignya phalerata Martius) is one of the most important palms in Brazil because of the largest morphological variation, wide geographic distribution, and high socio-economic importance. The diversity present in babassu germplasm should be protected against loss to ensure their use with high productivity. Study of the available variability in populations of babassu is necessary to develop conservation strategies. The study of genetic variability can be conducted using molecular markers and many of these studies require significant quantity of high-quality DNA. The present study aimed to effect comparison among eight DNA extraction methods in case of O. phalerata. The quality and concentration of nucleic acids were analyzed by spectrophotometry and integrity of DNA was ascertained by agarose gel electrophoresis. The spectrophotometry revealed that some methods resulted in high levels of concentration of nucleic acids, in which values of the ratio A260/280 and A260/230 were outside the range of purity. The agarose gel electrophoresis established the concentration and integrity of DNA. The methods of Murray and Thompson (1980) and Ferreira and Grattapaglia (1998) did not result in satisfactory quantities of DNA. Conversely, the method proposed by Khanuja et al. (1999) resulted in DNA of adequate quality and quantity that could be satisfactorily used for amplification reactions performed with two ISSR primers.

  11. DNA Vaccine Encoding HPV16 Oncogenes E6 and E7 Induces Potent Cell-mediated and Humoral Immunity Which Protects in Tumor Challenge and Drives E7-expressing Skin Graft Rejection.

    Science.gov (United States)

    Chandra, Janin; Dutton, Julie L; Li, Bo; Woo, Wai-Ping; Xu, Yan; Tolley, Lynn K; Yong, Michelle; Wells, James W; R Leggatt, Graham; Finlayson, Neil; Frazer, Ian H

    We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinical studies evaluating the efficacy of this vaccine in patients with HPV16 premalignancies are planned.

  12. DNA Vaccine Encoding HPV16 Oncogenes E6 and E7 Induces Potent Cell-mediated and Humoral Immunity Which Protects in Tumor Challenge and Drives E7-expressing Skin Graft Rejection

    Science.gov (United States)

    Chandra, Janin; Dutton, Julie L.; Li, Bo; Woo, Wai-Ping; Xu, Yan; Tolley, Lynn K.; Yong, Michelle; Wells, James W.; R. Leggatt, Graham; Finlayson, Neil

    2017-01-01

    We have previously shown that a novel DNA vaccine technology of codon optimization and the addition of ubiquitin sequences enhanced immunogenicity of a herpes simplex virus 2 polynucleotide vaccine in mice, and induced cell-mediated immunity when administered in humans at relatively low doses of naked DNA. We here show that a new polynucleotide vaccine using the same technology and encoding a fusion protein of the E6 and E7 oncogenes of high-risk human papillomavirus type 16 (HPV16) is immunogenic in mice. This vaccine induces long-lasting humoral and cell-mediated immunity and protects mice from establishment of HPV16-E7-expressing tumors. In addition, it suppresses growth of readily established tumors and shows enhanced efficacy when combined with immune checkpoint blockade targeted at PD-L1. This vaccine also facilitates rejection of HPV16-E7-expressing skin grafts that demonstrate epidermal hyperplasia with characteristics of cervical and vulvar intraepithelial neoplasia. Clinical studies evaluating the efficacy of this vaccine in patients with HPV16+ premalignancies are planned. PMID:28166181

  13. Identification of multiple HPV types on spermatozoa from human sperm donors

    DEFF Research Database (Denmark)

    Kaspersen, Maja D; Larsen, Peter B; Ingerslev, Hans Jakob

    2011-01-01

    Human papillomaviruses (HPV) may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV...... contain HPV, most of them of high-risk types binding to the equatorial segment of the sperm cell. Most HPV-positive sperm showed decreased staining with DAPI, indicative of reduced content of DNA. Our data demonstrate that oncogenic HPV types are frequent in men....... according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive...

  14. Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

    Science.gov (United States)

    Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

    2011-01-01

    Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

  15. HPV16 E6 seropositivity among cancer-free men with oral, anal or genital HPV16 infection.

    Science.gov (United States)

    Beachler, Daniel C; Waterboer, Tim; Pierce Campbell, Christine M; Ingles, Donna J; Kuhs, Krystle A Lang; Nyitray, Alan G; Hildesheim, Allan; Pawlita, Michael; Kreimer, Aimée R; Giuliano, Anna R

    2016-12-01

    Antibodies against the Human papillomavirus 16 (HPV16) E6 oncoprotein appear years prior to clinical diagnosis of anal and oropharyngeal cancer, but whether they develop around the time of HPV infection is unclear. Serum samples from 173 cancer-free men from the Human Papillomavirus Infection in Men (HIM) Study were tested for HPV antibodies and DNA. HPV16 E6 seropositivity was low among men with oral HPV16-infection (1/28; 3.6%, 95%CI=0.0%-18.4%), anal HPV16-infection (1/61; 1.6%, 95%CI=0.0%-8.8%), and 24-month persistent genital HPV16-infection (1/84; 1.2%, 0.0-6.5%). This suggests E6 seroconversion may not occur around the time of oral, anal, or genital HPV16 acquisition.

  16. Optimizing Fungal DNA Extraction Methods from Aerosol Filters

    Science.gov (United States)

    Jimenez, G.; Mescioglu, E.; Paytan, A.

    2016-12-01

    Fungi and fungal spores can be picked up from terrestrial ecosystems, transported long distances, and deposited into marine ecosystems. It is important to study dust-borne fungal communities, because they can stay viable and effect the ambient microbial populations, which are key players in biogeochemical cycles. One of the challenges of studying dust-borne fungal populations is that aerosol samples contain low biomass, making extracting good quality DNA very difficult. The aim of this project was to increase DNA yield by optimizing DNA extraction methods. We tested aerosol samples collected from Haifa, Israel (polycarbonate filter), Monterey Bay, CA (quartz filter) and Bermuda (quartz filter). Using the Qiagen DNeasy Plant Kit, we tested the effect of altering bead beating times and incubation times, adding three freeze and thaw steps, initially washing the filters with buffers for various lengths of time before using the kit, and adding a step with 30 minutes of sonication in 65C water. Adding three freeze/thaw steps, adding a sonication step, washing with a phosphate buffered saline overnight, and increasing incubation time to two hours, in that order, resulted in the highest increase in DNA for samples from Israel (polycarbonate). DNA yield of samples from Monterey (quart filter) increased about 5 times when washing with buffers overnight (phosphate buffered saline and potassium phophate buffer), adding a sonication step, and adding three freeze and thaw steps. Samples collected in Bermuda (quartz filter) had the highest increase in DNA yield from increasing incubation to 2 hours, increasing bead beating time to 6 minutes, and washing with buffers overnight (phosphate buffered saline and potassium phophate buffer). Our results show that DNA yield can be increased by altering various steps of the Qiagen DNeasy Plant Kit protocol, but different types of filters collected at different sites respond differently to alterations. These results can be used as

  17. Arduino-based automation of a DNA extraction system.

    Science.gov (United States)

    Kim, Kyung-Won; Lee, Mi-So; Ryu, Mun-Ho; Kim, Jong-Won

    2015-01-01

    There have been many studies to detect infectious diseases with the molecular genetic method. This study presents an automation process for a DNA extraction system based on microfluidics and magnetic bead, which is part of a portable molecular genetic test system. This DNA extraction system consists of a cartridge with chambers, syringes, four linear stepper actuators, and a rotary stepper actuator. The actuators provide a sequence of steps in the DNA extraction process, such as transporting, mixing, and washing for the gene specimen, magnetic bead, and reagent solutions. The proposed automation system consists of a PC-based host application and an Arduino-based controller. The host application compiles a G code sequence file and interfaces with the controller to execute the compiled sequence. The controller executes stepper motor axis motion, time delay, and input-output manipulation. It drives the stepper motor with an open library, which provides a smooth linear acceleration profile. The controller also provides a homing sequence to establish the motor's reference position, and hard limit checking to prevent any over-travelling. The proposed system was implemented and its functionality was investigated, especially regarding positioning accuracy and velocity profile.

  18. Optimization of DNA Extractions from Iron-rich Microbial Mats

    Science.gov (United States)

    Fullerton, H.; Hilton, T. S.; Moyer, C. L.

    2013-12-01

    Iron is the fourth most abundant element in the Earth's crust and is potentially one of the most abundant energy sources on the earth as an electron donor for chemolithoautotrophicgrowth coupled to Fe(II) oxidation. Many microbes have adapted to this energy source. One such bacterial class are the Zetaproteobacteria, which dominate Iron-rich microbial mats at Loihi seamount. Although cell counts are very high (up to 5.3x108 cells/ml), efficient DNA yields are low in comparison. In this study we compared extraction efficiency across different methods and with the addition of various buffers. Regardless of protocol (i.e., kit), the addition of sodium citrate drastically increased the DNA yield. The addition of sodium citrate did not alter community structure as determined by T-RFLP and qPCR. Citrate is a well-known ferric iron chelator and will bind ferrous as well. The chelated iron is then unable to participate in the Fenton reaction and this stops the generation of hydroxyl radicals which in turn can react and degrade the extracted DNA. We have utilized this relationship to allow us to obtain nearly an order of magnitude more microbial community DNA per sample, which should also have implications when processing low biomass samples, e.g., from the deep subsurface.

  19. DNA extraction from Ascaris suum muscle tissue

    OpenAIRE

    Di Mito, Carmela; Betschart, Bruno

    2009-01-01

    A new method for the extraction of DNA from Ascaris suum muscle has been developed. It combines a standard SDS-based extraction with a plant DNA extraction procedure. The use of SDS and proteinase K allows the elimination of proteins, while CTAB and polyclar AT eliminate glycogen and polyphenols. The DNA thus obtained can easily be digested by endonucleases and amplified by PCR.

  20. Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

    NARCIS (Netherlands)

    Vanysacker, L.; Declerck, S.A.J.; Hellemans, B.; De Meester, L.; Vankelecom, I.; Declerck, P.

    2010-01-01

    The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were

  1. Comparison of different protocols for the extraction of microbial DNA from reef corals

    NARCIS (Netherlands)

    Santos, H. F.; Carmo, F. L.; Leite, D. C. A.; Jesus, H. E.; De Carvalho Maalouf, P.; Almeida, C.; Soriano, A. U.; Altomari, D.; Suhett, L.; Volaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B. L.; Mendes, L. B.; Joao, R. R.; Lacava, B.; Jesus, R. F.; Sebastian, G.; Pessoa, A.; van Elsas, J. D.; Rezende, R. P.; Pires, D. O.; Duarte, G.; Castro, C. B.; Rosado, A. S.; Peixoto, R. S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR

  2. COMPARISON OF DIFFERENT PROTOCOLS FOR THE EXTRACTION OF MICROBIAL DNA FROM REEF CORALS

    NARCIS (Netherlands)

    Santos, H. F.; Carmo, F. L.; Leite, D. C. A.; Jesus, H. E.; De Carvalho Maalouf, P.; Almeida, C.; Soriano, A. U.; Altomari, D.; Suhett, L.; Volaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B. L.; Mendes, L. B.; Joao, R. R.; Lacava, B.; Jesus, R. F.; Sebastian, G.; Pessoa, A.; van Elsas, J. D.; Rezende, R. P.; Pires, D. O.; Duarte, G.; Castro, C. B.; Rosado, A. S.; Peixoto, R. S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR

  3. Comparison of different protocols for the extraction of microbial DNA from reef corals

    NARCIS (Netherlands)

    Santos, H. F.; Carmo, F. L.; Leite, D. C. A.; Jesus, H. E.; De Carvalho Maalouf, P.; Almeida, C.; Soriano, A. U.; Altomari, D.; Suhett, L.; Volaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B. L.; Mendes, L. B.; Joao, R. R.; Lacava, B.; Jesus, R. F.; Sebastian, G.; Pessoa, A.; van Elsas, J. D.; Rezende, R. P.; Pires, D. O.; Duarte, G.; Castro, C. B.; Rosado, A. S.; Peixoto, R. S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR

  4. Bacterial community analysis of activated sludge: an evaluation of four commonly used DNA extraction methods

    NARCIS (Netherlands)

    Vanysacker, L.; Declerck, S.A.J.; Hellemans, B.; De Meester, L.; Vankelecom, I.; Declerck, P.

    2010-01-01

    The effectiveness of three commercially available direct DNA isolation kits (Mobio, Fast, Qiagen) and one published direct DNA extraction protocol (Bead) for extracting bacterial DNA from different types of activated sludge was investigated and mutually compared. The DNA quantity and purity were det

  5. Assessing the Utility of Soil DNA Extraction Kits for Increasing DNA Yields and Eliminating PCR Inhibitors from Buried Skeletal Remains.

    Science.gov (United States)

    Hebda, Lisa M; Foran, David R

    2015-09-01

    DNA identification of human remains is often necessary when decedents are skeletonized; however, poor DNA recovery and polymerase chain reaction (PCR) inhibition are frequently encountered, a situation exacerbated by burial. In this research, the utility of integrating soil DNA isolation kits into buried skeletal DNA analysis was evaluated and compared to a standard human DNA extraction kit and organic extraction. The soil kits successfully extracted skeletal DNA at quantities similar to standard methods, although the two kits tested, which differ mechanistically, were not equivalent. Further, the PCR inhibitors calcium and humic acid were effectively removed using the soil kits, whereas collagen was less so. Finally, concordant control region sequences were obtained from human skeletal remains using all four methods. Based on these comparisons, soil DNA isolation kits, which quickened the extraction process, proved to be a viable extraction technique for skeletal remains that resulted in positive identification of a decedent. © 2015 American Academy of Forensic Sciences.

  6. HPV (Human Papillomavirus)

    Science.gov (United States)

    ... HPV? Yes. It tests for the kinds of HPV that may lead to cervical cancer. The FDA approved the HPV test to be ... number, but they will not turn into cancer. HPV and Cancer What should I know about cervical cancer? All women should get regular Pap tests. The ...

  7. Combination of treatment with death receptor 5-specific antibody with therapeutic HPV DNA vaccination generates enhanced therapeutic anti-tumor effects.

    Science.gov (United States)

    Tseng, Chih Wen; Monie, Archana; Trimble, Cornelia; Alvarez, Ronald D; Huh, Warner K; Buchsbaum, Donald J; Straughn, J Michael; Wang, Mei-Cheng; Yagita, Hideo; Hung, Chien-Fu; Wu, T-C

    2008-08-12

    There is currently a vital need for the development of novel therapeutic strategies for the control of advanced stage cancers. Antigen-specific immunotherapy and the employment of antibodies against the death receptor 5 (DR5) have emerged as two potentially promising strategies for cancer treatment. In the current study, we hypothesize that the combination of treatment with the anti-DR5 monoclonal antibody, MD5-1 with a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7(detox)) administered via gene gun would lead to further enhancement of E7-specific immune responses as well as anti-tumor effects. Our results indicated that mice bearing the E7-expressing tumor, TC-1 treated with MD5-1 monoclonal antibody followed by CRT/E7(detox) DNA vaccination generated the most potent therapeutic anti-tumor effects as well as highest levels of E7-specific CD8+ T cells among all the groups tested. In addition, treatment with MD5-1 monoclonal antibody was capable of rendering the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic T lymphocytes. Our findings serve as an important foundation for future clinical translation.

  8. Estudo epidemiológico do HPV na mucosa oral por meio de PCR Epidemiological study of HPV in oral mucosa through PCR

    Directory of Open Access Journals (Sweden)

    Willys Tristão

    2012-08-01

    Full Text Available Pertencente à família Papillomaviridae, o papilomavírus humano (HPV é formado por um capsídeo e uma única fita dupla de DNA. Sua infecção ocorre principalmente por forma sexual, apresentando grande tropismo por células cutâneas e mucosas. OBJETIVO: Avaliar a presença do HPV em mucosa oral de pacientes assintomáticos e em paralelo correlacionar os hábitos sociais comportamentais com a presença viral. MATERIAL E MÉTODO: Estudo de coorte contemporânea com corte transversal. O diagnóstico da presença viral foi realizado por PCR, utilizando os primers gerais MY09/11 em 125 amostras de mucosa oral, submetidas à extração de DNA e PCR para a pesquisa do gene da beta-globina para avaliação da qualidade do DNA extraído. Em paralelo, foi realizado um estudo de questões comportamentais dos pacientes. RESULTADOS: Todas as amostras apresentaram o diagnóstico positivo para o gene da betaglobina. O HPV foi diagnosticado em 23,2% das amostras analisadas. CONCLUSÃO: O vírus esteve presente em 29 dos 125 pacientes, sem que estes apresentassem qualquer manifestação clinicopatológica relacionada com o HPV. Quanto ao comportamento social dos pacientes, concluiu-se que a prática de sexo oral está correlacionada de forma estatisticamente significante com a presença viral, além de o HPV ter sido estatisticamente mais prevalente em pacientes do sexo feminino.The Human Papillomavirus (HPV belongs to the Papillomaviridae family and has a capsid and a single DNA strand. Its infection occurs mainly through sexual intercourse, having an important tropism for skin and mucosal cells. AIM: To evaluate the HPV presence in normal oral mucosa of asymptomatic subjects and; in parallel, to correlate social behavioral habits with the virus. MATERIALS AND METHODS: Contemporary cohort cross-sectional study. The HPV was found by PCR, using general primers MY09/11 in 125 oral mucosa samples submitted to DNA extraction and PCR to search for the beta

  9. An efficient and cost-effective method for DNA extraction from athalassohaline soil using a newly formulated cell extraction buffer.

    Science.gov (United States)

    Narayan, Avinash; Jain, Kunal; Shah, Amita R; Madamwar, Datta

    2016-06-01

    The present study describes the rapid and efficient indirect lysis method for environmental DNA extraction from athalassohaline soil by newly formulated cell extraction buffer. The available methods are mostly based on direct lysis which leads to DNA shearing and co-extraction of extra cellular DNA that influences the community and functional analysis. Moreover, during extraction of DNA by direct lysis from athalassohaline soil, it was observed that, upon addition of poly ethylene glycol (PEG), isopropanol or absolute ethanol for precipitation of DNA, salt precipitates out and affecting DNA yield significantly. Therefore, indirect lysis method was optimized for extraction of environmental DNA from such soil containing high salts and low microbial biomass (CFU 4.3 × 10(4) per gram soil) using newly formulated cell extraction buffer in combination with low and high speed centrifugation. The cell extraction buffer composition and its concentration were optimized and PEG 8000 (1 %; w/v) and 1 M NaCl gave maximum cell mass for DNA extraction. The cell extraction efficiency was assessed with acridine orange staining of soil samples before and after cell extraction. The efficiency, reproducibility and purity of extracted DNA by newly developed procedure were compared with previously recognized methods and kits having different protocols including indirect lysis. The extracted environmental DNA showed better yield (5.6 ± 0.7 μg g(-1)) along with high purity ratios. The purity of DNA was validated by assessing its usability in various molecular techniques like restriction enzyme digestion, amplification of 16S rRNA gene using PCR and UV-Visible spectroscopy analysis.

  10. Evaluation of four automated protocols for extraction of DNA from FTA cards.

    Science.gov (United States)

    Stangegaard, Michael; Børsting, Claus; Ferrero-Miliani, Laura; Frank-Hansen, Rune; Poulsen, Lena; Hansen, Anders J; Morling, Niels

    2013-10-01

    Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

  11. HPV in men.

    Science.gov (United States)

    D'Hauwers, K W M; Tjalma, W A A

    2008-01-01

    To collect information about HPV in men and the (possible) correlation with HVP infection in women. Review of the literature. An overview of HPV-related penile and anal malignancies in men and the risk factors of acquiring HPV. In men HPV is also partially responsible for anogenital malignancies. Although the prevalence of HPV-related malignancies in men is much lower than in women, it is useful to gain more knowlege. Especially knowing if men are really the HPV reservoir and transmitters for women can make a difference in deciding whether men should also be screened for HPV and if they are good candidates for vaccination.

  12. Comparison of four commercial DNA extraction kits for the recovery of Bacillus spp. spore DNA from spiked powder samples.

    Science.gov (United States)

    Mölsä, Markos; Kalin-Mänttäri, Laura; Tonteri, Elina; Hemmilä, Heidi; Nikkari, Simo

    2016-09-01

    Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. A single protocol for extraction of gDNA from bacteria and yeast.

    Science.gov (United States)

    Vingataramin, Laurie; Frost, Eric H

    2015-03-01

    Guanidine thiocyanate breakage of microorganisms has been the standard initial step in genomic DNA (gDNA) extraction of microbial DNA for two decades, despite the requirement for pretreatments to extract DNA from microorganisms other than Gram-negative bacteria. We report a quick and low-cost gDNA extraction protocol called EtNa that is efficient for bacteria and yeast over a broad range of concentrations. EtNa is based on a hot alkaline ethanol lysis. The solution can be immediately centrifuged to yield a crude gDNA extract suitable for PCR, or it can be directly applied to a silica column for purification.

  14. Seroconversion Following Anal and Genital HPV Infection in Men: The HIM Study

    Science.gov (United States)

    Giuliano, Anna R.; Viscidi, Raphael; Torres, B. Nelson; Ingles, Donna J.; Sudenga, Staci L.; Villa, Luisa L.; Baggio, Maria Luiza; Abrahamsen, Martha; Quiterio, Manuel; Salmeron, Jorge; Lazcano-Ponce, Eduardo

    2015-01-01

    Background Protection from naturally acquired human papillomavirus (HPV) antibodies may influence HPV infection across the lifespan. This study describes seroconversion rates following genital, anal, and oral HPV 6/11/16/18 infections in men and examines differences by HPV type and anatomic site. Methods Men with HPV 6/11/16/18 infections who were seronegative for those genotypes at the time of DNA detection were selected from the HPV Infection in Men (HIM) Study. Sera specimens collected ≤36 months after detection were analyzed for HPV 6/11/16/18 antibodies using a virus-like particle-based ELISA. Time to seroconversion was separately assessed for each anatomic site, stratified by HPV type. Results Seroconversion to ≥1 HPV type (6/11/16/18) in this sub-cohort (N=384) varied by anatomic site, with 6.3, 18.9, and 0.0% seroconverting following anal, genital, and oral HPV infection, respectively. Regardless of anatomic site, seroconversion was highest for HPV 6 (19.3%). Overall, seroconversion was highest following anal HPV 6 infection (69.2%). HPV persistence was the only factor found to influence seroconversion. Conclusions Low seroconversion rates following HPV infection leave men susceptible to recurrent infections that can progress to HPV-related cancers. This emphasizes the need for HPV vaccination in men to ensure immune protection against new HPV infections and subsequent disease. PMID:26688833

  15. DNA extraction on bio-chip: history and preeminence over conventional and solid-phase extraction methods.

    Science.gov (United States)

    Ayoib, Adilah; Hashim, Uda; Gopinath, Subash C B; Md Arshad, M K

    2017-09-23

    This review covers a developmental progression on early to modern taxonomy at cellular level following the advent of electron microscopy and the advancement in deoxyribonucleic acid (DNA) extraction for expatiation of biological classification at DNA level. Here, we discuss the fundamental values of conventional chemical methods of DNA extraction using liquid/liquid extraction (LLE) followed by development of solid-phase extraction (SPE) methods, as well as recent advances in microfluidics device-based system for DNA extraction on-chip. We also discuss the importance of DNA extraction as well as the advantages over conventional chemical methods, and how Lab-on-a-Chip (LOC) system plays a crucial role for the future achievements.

  16. Designing easy DNA extraction: Teaching creativity through laboratory practice.

    Science.gov (United States)

    Susantini, Endang; Lisdiana, Lisa; Isnawati; Tanzih Al Haq, Aushia; Trimulyono, Guntur

    2017-05-01

    Subject material concerning Deoxyribose Nucleic Acid (DNA) structure in the format of creativity-driven laboratory practice offers meaningful learning experience to the students. Therefore, a laboratory practice in which utilizes simple procedures and easy-safe-affordable household materials should be promoted to students to develop their creativity. This study aimed to examine whether designing and conducting DNA extraction with household materials could foster students' creative thinking. We also described how this laboratory practice affected students' knowledge and views. A total of 47 students participated in this study. These students were grouped and asked to utilize available household materials and modify procedures using hands-on worksheet. Result showed that this approach encouraged creative thinking as well as improved subject-related knowledge. Students also demonstrated positive views about content knowledge, social skills, and creative thinking skills. This study implies that extracting DNA with household materials is able to develop content knowledge, social skills, and creative thinking of the students. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):216-225, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

  17. Comparison of the immunogenicity of Cervarix® and Gardasil® human papillomavirus vaccines for oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults.

    Science.gov (United States)

    Toft, Lars; Tolstrup, Martin; Müller, Martin; Sehr, Peter; Bonde, Jesper; Storgaard, Merete; Østergaard, Lars; Søgaard, Ole S

    2014-01-01

    Individuals infected with human immunodeficiency virus (HIV) have excess risk of developing human papillomavirus (HPV)-related disease. A substantial fraction of HPV-associated cancers is caused by HPV serotypes not included in the currently available vaccines. Among healthy women, both Cervarix(®) (HPV-16/18, GlaxoSmithKline Biologicals, GSK) and Gardasil(®) (HPV-6/11/16/18, Merck) have demonstrated partial cross-protection against certain oncogenic non-vaccine HPV-types. Currently, there are no available data on vaccine-induced cross-protection in men and little is known about cross-reactive immunity after HPV-vaccination of HIV-infected individuals. In an investigator-initiated trial, we randomized 91 HIV-positive men and women to receive vaccination with Cervarix(®) or Gardasil(®). The HPV-DNA status of the participants was determined with pcr before and after immunization. Cross-reactive antibody responses against HPV-31, HPV-33, and HPV-45 were evaluated for up to 12 months using a pseudovirion-based neutralization assay (PBNA). Geometric mean antibody titers (GMTs) were compared among vaccine groups and genders at 7 and 12 months.: Both vaccines induced anti-HPV-31, -33, and -45 neutralizing antibodies in participants who were seronegative and HPV-DNA negative for those types at study entry. Geometric mean antibody titers were comparable between vaccine groups. Interestingly, anti-HPV-31 and -33 antibody titers were higher among women compared with men at 7 and 12 months.: In conclusion, both licensed HPV-vaccines induced cross-neutralizing antibodies against frequent oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults, and women had greater serological responses against HPV-31 and -33 compared with men.

  18. Can human papillomavirus (HPV) genotyping classify non-16/18 high-risk HPV infection by risk stratification?

    Science.gov (United States)

    Sung, Yeoun Eun; Ki, Eun Young; Lee, Youn Soo; Hur, Soo Young; Lee, Ahwon; Park, Jong Sup

    2016-11-01

    Infection with high-risk genotypes of human papillomavirus (HR-HPV) is the major cause of invasive cervical cancers. HPV-16 and HPV-18 are known to be responsible for two-thirds of all invasive cervical carcinomas, followed by HPV-45, -31, and -33. Current guidelines only differentiate HPV-16/18 (+) by recommending direct colposcopy for treatment. We tried to evaluate whether there are differences in risk among 12 non-16/18 HR-HPV genotypes in this study. The pathology archive database records of 1,102 consecutive gynecologic patients, who had results for cervical cytology and histology and for HPV testing, as determined by HPV 9G DNA chip, were reviewed. Among the 1,102 patients, 346 were non-16/18 HR-HPV (+) and 231 were HPV-16/18 (+). We calculated the odds ratios for ≥cervical intraepithelial neoplasia 2 (CIN 2) of 14 groups of each HR-HPV genotype compared with a group of HR-HPV (-) patients. Based on the odds ratio of each genotype, we divided patients with non-16/18 HR-HPV genotypes (+) into two groups: HPV-31/33/35/45/52/58 (+) and HPV-39/51/56/59/66/68 (+). The age-adjusted odds ratios for ≥CIN 2 of the HPV-31/33/35/45/52/58 (+) and HPV-39/51/56/59/66/68 (+) groups compared with a HR-HPV (-) group were 11.9 (95% CI, 7.6 to 18.8; p<0.001) and 2.4 (95% CI, 1.4 to 4.3; p<0.001), respectively, while that of the HPV-16/18 (+) group was 18.1 (95% CI, 11.6 to 28.3; p=0.003). The 12 non-16/18 HR-HPV genotypes can be further categorized (HPV-31/33/35/45/52/58 vs. HPV-39/51/56/59/66/68) by risk stratification. The HPV-31/33/35/45/52/58 genotypes might need more aggressive action. Large scale clinical trials or cohort studies are necessary to confirm our suggestion.

  19. Factors associated with HPV-DNA clearance in a cohort of HIV-positive patients: role of cART and gender

    Directory of Open Access Journals (Sweden)

    Elisa Suardi

    2014-11-01

    Full Text Available Introduction: We aimed to assess any factors associated with dysplasia regression and with HPV clearance in a cohort of HIV+ patients, with particular focus on cART and gender. Methods: Asymptomatic HIV+ patients of the San Paolo Infectious Disease (SPID cohort who underwent anoscopy/gynaecological evaluation were enrolled. Anal/cervical brushing were analyzed for: HPV-PCR detection/genotyping (HR-HPV, cytologic abnormalities (Bethesda System 2001: LSIL-HSIL. Demographics and HIV-related parameters were evaluated at baseline. Activated CD8+/CD38+ lymphocytes were measured (flow citometry. Patients were examined at baseline (T0 and at 12–18 months visit (T1. HPV clearance was defined as negativisation of HPV at T1; SIL regression (SIL-R and progression (SIL-P were defined as change from HSIL/LSIL to a lower-grade/absence of dysplasia and as change from absence of HSIL/LSIL to a higher-grade dysplasia at T1, respectively. Mann Whitney test, Chi-square test and multivariate logistic regression were used. Results: A total of 189 patients were examined, 60 (32% were women. One hundred fifty patients (79% were HPV+, 113 (75% harboured HR-HPV; 103 (68% showed LSIL/HSIL at T0 (32% of women and 65% of men (all were HPV-positive. No differences in demographics and HIV-related markers were found between patients with SIL-P (33, 41% and patients with SIL-R (47, 59%. HPV+ patients who cleared HPV (28, 18% were found to be more frequently female, heterosexual infected, more frequently on cART and with lower Log10 HIV-RNA and lower levels of CD8+/CD38+ % compared with HPV persistence group (Table 1.No differences in PI exposure were found between the two groups (p=.08. Interestingly, also when only HR-HPV were considered, clearance was associated with exposure to cART (naïve 4%, vs cART 86%, p=.048. In multivariate analysis, heterosexuals (AOR 5.123, 95% CI 1.5–17.5 vs homosexuals were independently associated to HPV clearance, whereas CD8+/CD38+% (AOR 0

  20. Factors associated with HPV-DNA clearance in a cohort of HIV-positive patients: role of cART and gender

    Science.gov (United States)

    Suardi, Elisa; Bai, Francesca; Comi, Laura; Pandolfo, Alessandro; Rovati, Marco; Barco, Ambra; Dalzero, Serena; Cassani, Barbara; Marchetti, Giulia; D'Arminio Monforte, Antonella

    2014-01-01

    Introduction We aimed to assess any factors associated with dysplasia regression and with HPV clearance in a cohort of HIV+ patients, with particular focus on cART and gender. Methods Asymptomatic HIV+ patients of the San Paolo Infectious Disease (SPID) cohort who underwent anoscopy/gynaecological evaluation were enrolled. Anal/cervical brushing were analyzed for: HPV-PCR detection/genotyping (HR-HPV), cytologic abnormalities (Bethesda System 2001: LSIL-HSIL). Demographics and HIV-related parameters were evaluated at baseline. Activated CD8+/CD38+ lymphocytes were measured (flow citometry). Patients were examined at baseline (T0) and at 12–18 months visit (T1). HPV clearance was defined as negativisation of HPV at T1; SIL regression (SIL-R) and progression (SIL-P) were defined as change from HSIL/LSIL to a lower-grade/absence of dysplasia and as change from absence of HSIL/LSIL to a higher-grade dysplasia at T1, respectively. Mann Whitney test, Chi-square test and multivariate logistic regression were used. Results A total of 189 patients were examined, 60 (32%) were women. One hundred fifty patients (79%) were HPV+, 113 (75%) harboured HR-HPV; 103 (68%) showed LSIL/HSIL at T0 (32% of women and 65% of men) (all were HPV-positive). No differences in demographics and HIV-related markers were found between patients with SIL-P (33, 41%) and patients with SIL-R (47, 59%). HPV+ patients who cleared HPV (28, 18%) were found to be more frequently female, heterosexual infected, more frequently on cART and with lower Log10 HIV-RNA and lower levels of CD8+/CD38+ % compared with HPV persistence group (Table 1). No differences in PI exposure were found between the two groups (p=.08). Interestingly, also when only HR-HPV were considered, clearance was associated with exposure to cART (naïve 4%, vs cART 86%, p=.048). In multivariate analysis, heterosexuals (AOR 5.123, 95% CI 1.5–17.5 vs homosexuals) were independently associated to HPV clearance, whereas CD8+/CD38+% (AOR 0

  1. AN UPWARD TREND IN DNA P16INK4A METHYLATION PATTERN AND HIGH RISK HPV INFECTION ACCORDING TO THE SEVERITY OF THE CERVICAL LESION

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    Fernanda Nahoum Carestiato

    2013-09-01

    Full Text Available SUMMARY High-risk human papillomavirus (hr-HPV infection is necessary but not sufficient for cervical cancer development. Recently, P16INK4A gene silencing through hypermethylation has been proposed as an important cofactor in cervical carcinogenesis due to its tumor suppressor function. We aimed to investigate P16INK4A methylation status in normal and neoplastic epithelia and evaluate an association with HPV infection and genotype. This cross-sectional study was performed with 141 cervical samples from patients attending Hospital Moncorvo Filho, Rio de Janeiro. HPV detection and genotyping were performed through PCR and P16INK4A methylation by nested-methylation specific PCR (MSP. HPV frequency was 62.4% (88/141. The most common HPV were HPV16 (37%, HPV18 (16.3% and HPV33/45(15.2%. An upward trend was observed concerning P16INK4A methylation and lesion degree: normal epithelia (10.7%, low grade lesions (22.9%, high grade (57.1% and carcinoma (93.1% (p < 0.0001. A multivariate analysis was performed to evaluate an association between methylation, age, tobacco exposure, HPV infection and genotyping. A correlation was found concerning methylation with HPV infection (p < 0.0001, hr-HPV (p = 0.01, HSIL (p < 0.0007 and malignant lesions (p < 0.0001. Since viral infection and epigenetic alterations are related to cervical carcinoma, we suggest that P16INK4A methylation profile maybe thoroughly investigated as a biomarker to identify patients at risk of cancer.

  2. Human Papillomavirus (HPV infection in pregnant women and mother-to-child transmission of genital HPV genotypes: a prospective study in Spain

    Directory of Open Access Journals (Sweden)

    Albero Ginesa

    2009-05-01

    Full Text Available Abstract Background Studies on HPV infection in pregnant women and HPV transmission to the child have yielded inconsistent results. Methods To estimate mother-to-child HPV transmission we carried out a prospective cohort study that included 66 HPV-positive and 77 HPV-negative pregnant women and their offspring attending a maternity hospital in Barcelona. To estimate HPV prevalence and genotype distribution in pregnancy we also carried out a related screening survey of cervical HPV-DNA detection among 828 pregnant women. Cervical cells from the mother were collected at pregnancy (mean of 31 weeks and at the 6-week post-partum visit. Exfoliated cells from the mouth and external genitalia of the infants were collected around birth, at the 6-week post-partum visit, and around 3, 6, 12, and 24 months of age. All samples were tested for HPV using PCR. Associations between potential determinants of HPV infection in pregnant women and of HPV positivity in infants were also explored by logistic regression modelling. Results Overall cervical HPV-DNA detection in pregnant women recruited in the HPV screening survey was 6.5% (54/828. Sexual behavior-related variables, previous histories of genital warts or sexually transmitted infections, and presence of cytological abnormalities were statistically significantly and positively associated with HPV DNA detection in pregnant women recruited in the cohort. At 418 infant visits and a mean follow-up time of 14 months, 19.7% of infants born to HPV-positive mothers and 16.9% of those born to HPV-negative mothers tested HPV positive at some point during infants' follow-up. The most frequently detected genotype both in infants and mothers was HPV-16, after excluding untyped HPV infections. We found a strong and statistically significant association between mother's and child's HPV status at the 6-week post-partum visit. Thus, children of mothers' who were HPV-positive at the post-partum visit were about 5 times more

  3. [Genomic DNA extraction from hair sacs of pigs using modified phenol-chloroform method].

    Science.gov (United States)

    Wang, Ji-Ying; Yu, Ying; Feng, Li-Xia; Wang, Huai-Zhong; Zhang, Qin

    2010-07-01

    In referring to various methods for genomic DNA extraction from different tissues, we modified the classical phenol-chloroform procedure and reaction system for use in genomic DNA extraction from pig hair sacs. With the modified the phenol-chloroform method we successfully obtained high quality genomic DNA from pig hair sacs. Genomic DNA can be extracted from sacs of one to six pig hairs with satisfied quantity and quality for the need of PCR-based molecular ex-periment.

  4. Comparing two protocols of DNA extraction of Trypanosoma cruzi cultured in axenic medium

    OpenAIRE

    López, Mariela; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Escuela de Bioanálisis, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en Bioanálisis; Rivera, María G.; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en Bioanálisis.; Viettri, Mercedes; Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en Bioanálisis.; Lares, María; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Técnico superior universitario en Química.; Morocoima, Antonio; Centro de Medicina Tropical de Oriente, Facultad de Medicina, Universidad de Oriente. Anzoátegui, Venezuela. Médico cirujano, magíster en Parasitología.; Herrera, Leidi; Instituto de Zoología y Ecología Tropical, Facultad de Ciencias, Universidad Central de Venezuela. Caracas, Venezuela. Biólogo, doctora en Ciencias.; Ferrer, Elizabeth; Instituto de Investigaciones Biomédicas “Dr. Francisco J. Triana Alonso”, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Departamento de Parasitología, Facultad de Ciencias de la Salud, Universidad de Carabobo. Maracay, Venezuela. Licenciada en bioanálisis, doctora en Biología Molecular.

    2014-01-01

    Objectives. To compare two extraction protocols of Trypanosoma cruzi DNA for use in DNA amplification of kinetoplast minicircles (kDNA) through the technique of Polymerase Chain Reaction (PCR). Materials and methods. Epimastigotes of T. cruzi were cultured in axenic conditions and masses from 1.5 to 100 x 106 parasites were obtained. DNA extraction was performed using two protocols: extraction with organic solvents (phenol/chloroform), and with resin (Chelex®100), from different parasitic sed...

  5. HPV E6/E7 mRNA检测对宫颈癌筛查意义的初步评价%Comparative application of HPV E6/E7 mRNA and DNA test used in cervical cancer screening of Ningde aera

    Institute of Scientific and Technical Information of China (English)

    黄宝英; 周伦顺; 富显果; 余兰; 卢少玲; 陈惠华; 缪韵仪; 刘桐宇; lulu zhang

    2013-01-01

    OBJECTIVE:To investigate the role of testing E6/E7 mRNA of high risk human papilloma virus (HPV) directly from liquid-based thin layer cytology test (LCT) samples for cervical cancer screening.METHODS:Branched DNA (bDNA) technology was used to detect high risk HPV E6/E7 mRNA and DNA directly from LCT samples.The data according to cytological grades or pathology were statistically analyzed.RESULTS:We demonstrated the detectable rate of high risk HPV E6/E7 mRNA and high risk HPV E6/E7 DNA in all 5 cytological grades among 231 samples with chi square test.Positive detection rate difference was not significant in inflammation or normal cell group (78 cases,x2=2.307,P>0.05).IN ASCUS group(140 cases),positive detection rate difference was statistically significant (x2 =9.082,P=0.020).Positive detection rate difference was not significant in LSIL (n =8) or HSIL (n =5) group (P value were 0.464 and 0.800).And 34 cases of ASCUS specimens were verified by pathology.The detectable rate of high risk E6/E7 mRNA test was equal to the test of high risk HPV E6/E7 DNA in normal/inflammation or LSIL/HSIL group (P>0.05).The detectable rate of high risk HPV E6/E7 mRNA in all pathological grades among 43 samples was almost identical to high risk HPV E6/E7 DNA (P>0.05).The sensitivity of high risk HPV E6/E7 mRNA test was 33.33% (95%CI=17.19-54.63).The specificity of high risk HPV E6/E7 mRNA test was 72.73%(95%CI=51.85-86.85),while positive predictive value (PPV) was 53.85 % (95 %CI=29.14-76.79),and negative predictive value (NPV) was 53.33 % (95 %CI=36.14-69.77),respectively.The high risk HPV E6/E7 mRNA test associated with LCT test may enhance clinical sensitivity (100%,95% CI =84.54-100.00) and negative predictive value (100%,95% CI =43.85-100.00) in cervical cancer screening.CONCLUSIONS:The tests of high risk HPV E6/E7 mRNA can be a useful tool for cervical cancer screening.Applying E6/E7 mRNA test combined with LCT may be more effective in screening and

  6. Extraction of DNA from malaria-infected erythrocytes using isotachophoresis.

    Science.gov (United States)

    Marshall, Lewis A; Han, Crystal M; Santiago, Juan G

    2011-12-15

    We demonstrate a technique for purification of nucleic acids from malaria parasites infecting human erythrocytes using isotachophoresis (ITP). We release nucleic acids from malaria-infected erythrocytes by lysing with heat and proteinase K for 10 min and immediately, thereafter, load sample onto a capillary device. We study the effect of temperature on lysis efficiency. We also implement pressure-driven counterflow during ITP extraction to extend focusing time and increase nucleic acid yield. We show that the purified genomic DNA samples are compatible with polymerase chain reaction (PCR) and demonstrate a clinically relevant limit of detection of 0.5 parasites per nanoliter using quantitative PCR.

  7. Determination of an efficient and reliable method for DNA extraction from ticks.

    Science.gov (United States)

    Halos, Lénaïg; Jamal, Taoufik; Vial, Laurence; Maillard, Renaud; Suau, Antonia; Le Menach, Arnaud; Boulouis, Henri-Jean; Vayssier-Taussat, Muriel

    2004-01-01

    Molecular detection of pathogenic microorganisms in ticks is based on DNA amplification of the target pathogen; therefore, extraction of DNA from the tick is a major step. In this study, we compared three different tick DNA extraction protocols based on an enzymatic digestion by proteinase K followed by DNA extraction by a commercial kit (method 1), or on mortar crushing, proteinase K digestion and phenol/chloroform DNA extraction (method 2) and fine crushing with a beads beater, proteinase K digestion and DNA extraction using a commercial kit (method 3). The absence of PCR inhibitors and the DNA quality were evaluated by PCR amplification of the tick mitochondrial 16S rRNA gene using tick-specific primers. With method 1, 23/30 (77%) of the samples were extracted; with method 2, 30/31 (97%) of the samples were extracted and with method 3, 30/30 (100%) of the samples were extracted. DNA extraction efficiency using method 3 is significantly higher than DNA extraction efficiency using method 1 (100% versus 77%, P DNA extraction and applicable to the treatment of small samples such as nymphs and soft ticks with 100% efficiency.

  8. HPV infection among rural American Indian women and urban white women in South Dakota: an HPV prevalence study

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    Muller Clemma J

    2011-09-01

    Full Text Available Abstract Background High-risk strains of human papillomavirus (HPV cause cervical cancer. American Indian (AI women in the Northern Plains of the U.S. have significantly higher incidence and mortality rates for cervical cancer than White women in the same geographical area. We compared HPV prevalence, patterns of HPV types, and infection with multiple HPV types in AI and White women living in South Dakota, U.S. Methods We analyzed the HPV status of cervical samples collected in 2006-2008 from women aged 18-65 years who attended two rural AI reservation clinics (n = 235 or an urban clinic in the same area serving mostly White women (n = 246. Data collection occurred before HPV vaccination was available to study participants. HPV DNA was amplified by using the L1 consensus primer system and an HPV Linear Array detection assay to identify HPV types. We used chi-square tests to compare HPV variables, with percentages standardized by age and lifetime number of sexual partners. Results Compared to White women, AI women were younger (p = 0.01 and reported more sexual partners (p p p = 0.001. Infections among AI women showed a wider variety and very different pattern of HPV types, including a higher prevalence of mixed HPV infections (19% [95% CI = 26-38] vs. 7% [95% CI = 4-11]; p = 0.001. AI women had a higher percentage of HPV infections that were not preventable by HPV vaccination (32% [95% CI = 26-38] vs. 15% [95% CI = 11-21]; p Conclusions A higher HPV burden and a different HPV genotyping profile may contribute to the high rate of cervical cancer among AI women.

  9. Human papilloma virus (HPV) molecular diagnostics.

    Science.gov (United States)

    Kroupis, Christos; Vourlidis, Nikolaos

    2011-11-01

    Human Papilloma Virus (HPV) is becoming a menace worldwide, especially to the developing world, due to its involvement in a variety of malignancies, with cervical cancer being the most important and prevalent. There are many HPV types; HPV 16/18 are the most carcinogenic but few others are also characterized as high-risk (HR). They can cause a variety of low- or high-grade cellular abnormalities, most frequently detected in a routine Pap test. Most infections clear within 2 years, however, a minority persists and potentially could progress to cervical cancer. Molecular tests detecting HPV DNA, RNA or proteins are now being available either commercially or in-house developed. DNA detection is nowadays an established tool for diagnosis and monitoring of HPV-related disease, however, there is lack of a reference method and standardization with reference materials. The various available test formats create confusion on which molecular test to choose and what are its limitations. Therefore, the need for lab accreditation and participation in proficiency testing has to be stressed. Novel HPV biomarkers (RNA, protein etc.) are now intensively examined for their inclusion as adjunct tools. Recently, developed prophylactic vaccines for HPV 16/18 have already proven safe and efficient and raise high expectations for the complete eradication of these types in the future.

  10. Comparison of the immunogenicity of Cervarix(®) and Gardasil(®) human papillomavirus vaccines for oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults

    DEFF Research Database (Denmark)

    Toft, Lars; Tolstrup, Martin; Müller, Martin

    2014-01-01

    -reactive antibody responses against HPV-31, HPV-33, and HPV-45 were evaluated for up to 12 months using a pseudovirion-based neutralization assay (PBNA). Geometric mean antibody titers (GMTs) were compared among vaccine groups and genders at 7 and 12 months. Both vaccines induced anti-HPV-31, -33, and -45...... neutralizing antibodies in participants who were seronegative and HPV-DNA negative for those types at study entry. Geometric mean antibody titers were comparable between vaccine groups. Interestingly, anti-HPV-31 and -33 antibody titers were higher among women compared with men at 7 and 12 months......Individuals infected with human immunodeficiency virus (HIV) have excess risk of developing human papillomavirus (HPV)-related disease. A substantial fraction of HPV-associated cancers is caused by HPV serotypes not included in the currently available vaccines. Among healthy women, both Cervarix...

  11. Use of HPV testing for cervical screening in vaccinated women--Insights from the SHEVa (Scottish HPV Prevalence in Vaccinated Women) study.

    Science.gov (United States)

    Bhatia, Ramya; Kavanagh, Kimberley; Cubie, Heather Ann; Serrano, Itziar; Wennington, Holli; Hopkins, Mark; Pan, Jiafeng; Pollock, Kevin G; Palmer, Tim J; Cuschieri, Kate

    2016-06-15

    The management of cervical disease is changing worldwide as a result of HPV vaccination and the increasing use of HPV testing for cervical screening. However, the impact of vaccination on the performance of HPV based screening strategies is unknown. The SHEVa (Scottish HPV Prevalence in Vaccinated women) projects are designed to gain insight into the impact of vaccination on the performance of clinically validated HPV assays. Samples collated from women attending for first cervical smear who had been vaccinated as part of a national "catch-up" programme were tested with three clinically validated HPV assays (2 DNA and 1 RNA). Overall HR-HPV and type specific positivity was assessed in total population and according to underlying cytology and compared to a demographically equivalent group of unvaccinated women. HPV prevalence was significantly lower in vaccinated women and was influenced by assay-type, reducing by 23-25% for the DNA based assays and 32% for the RNA assay (p = 0.0008). All assays showed over 75% reduction of HPV16 and/or 18 (p HPV was not significantly different in vaccinated vs unvaccinated women. In women with low grade abnormalities, the proportion associated with non 16/18 HR-HPV was significantly higher in vaccinated women (p HPV assays are affected differentially when applied to vaccinated women, dependent on assay chemistry. The increased proportion of non HPV16/18 infections may have implications for clinical performance, consequently, longitudinal studies linking HPV status to disease outcomes in vaccinated women are warranted.

  12. DNA extraction from sea anemone (Cnidaria: Actiniaria tissues for molecular analyses

    Directory of Open Access Journals (Sweden)

    Pinto S.M.

    2000-01-01

    Full Text Available A specific DNA extraction method for sea anemones is described in which extraction of total DNA from eight species of sea anemones and one species of corallimorpharian was achieved by changing the standard extraction protocols. DNA extraction from sea anemone tissue is made more difficult both by the tissue consistency and the presence of symbiotic zooxanthellae. The technique described here is an efficient way to avoid problems of DNA contamination and obtain large amounts of purified and integral DNA which can be used in different kinds of molecular analyses.

  13. An efficient method for genomic DNA extraction from different molluscs species.

    Science.gov (United States)

    Pereira, Jorge C; Chaves, Raquel; Bastos, Estela; Leitão, Alexandra; Guedes-Pinto, Henrique

    2011-01-01

    The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills) or quantity of tissue, can explain the lack of efficiency (quality and yield) in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia) and Muricidae (Gastropoda), with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others.

  14. An Efficient Method for Genomic DNA Extraction from Different Molluscs Species

    Directory of Open Access Journals (Sweden)

    Henrique Guedes-Pinto

    2011-11-01

    Full Text Available The selection of a DNA extraction method is a critical step when subsequent analysis depends on the DNA quality and quantity. Unlike mammals, for which several capable DNA extraction methods have been developed, for molluscs the availability of optimized genomic DNA extraction protocols is clearly insufficient. Several aspects such as animal physiology, the type (e.g., adductor muscle or gills or quantity of tissue, can explain the lack of efficiency (quality and yield in molluscs genomic DNA extraction procedure. In an attempt to overcome these aspects, this work describes an efficient method for molluscs genomic DNA extraction that was tested in several species from different orders: Veneridae, Ostreidae, Anomiidae, Cardiidae (Bivalvia and Muricidae (Gastropoda, with different weight sample tissues. The isolated DNA was of high molecular weight with high yield and purity, even with reduced quantities of tissue. Moreover, the genomic DNA isolated, demonstrated to be suitable for several downstream molecular techniques, such as PCR sequencing among others.

  15. Comparison of DNA extraction methods for detection of citrus huanglongbing in Colombia

    Directory of Open Access Journals (Sweden)

    Jorge Evelio Ángel

    2014-04-01

    Full Text Available Four DNA citrus plant tissue extraction protocols and three methods of DNA extraction from vector psyllid Diaphorina citri Kuwayama (Hemiptera: Psyllidae were compared as part of the validation process and standardization for detection of huanglongbing (HLB. The comparison was done using several criterias such as integrity, purity and concentration. The best quality parameters presented in terms of extraction of DNA from plant midribs tissue of citrus, were cited by Murray and Thompson (1980 and Rodríguez et al. (2010, while for the DNA extraction from psyllid vectors of HLB, the best extraction method was suggested by Manjunath et al.(2008.

  16. Establishment and application of an efficient, economic, and rapid rice DNA extraction method

    Science.gov (United States)

    A rapid, economic, and efficient method for DNA extraction from rice leaf, root and seed was developed, and the extracted DNA was used as a template to successfully amplify the rice blast resistance gene Pi-ta. Profiles of Pi-ta in 165 breeding lines detected by DNA markers were verified using diff...

  17. Highly Effective DNA Extraction Method from Fresh, Frozen, Dried and Clotted Blood Samples

    Directory of Open Access Journals (Sweden)

    Jaleh Barar

    2011-09-01

    Full Text Available Introduction: Today, with the tremendous potential of genomics and other recent advances in science, the role of science to improve reliable DNA extraction methods is more relevant than ever before. The ideal process for genomic DNA extraction demands high quantities of pure, integral and intact genomic DNA (gDNA from the sample with minimal co-extraction of inhibitors of downstream processes. Here, we report the development of a very rapid, less-hazardous, and high throughput protocol for extracting of high quality DNA from blood samples. Methods: Dried, clotted and ethylene diamine tetra-acetic acid (EDTA treated fresh and frozen blood samples were extracted using this method in which the quality and integrity of the extracted DNA were corroborated by agarose gel electrophoresis, PCR reaction and DNA digestion using restricted enzyme. The UV spectrophotometric and gel electrophoresis analysis resulted in high A260/A280 ratio (>1.8 with high intactness of DNA. Results: PCR and DNA digestion experiments indicated that the final solutions of extracted DNA contained no inhibitory substances, which confirms that the isolated DNA is of good quality. Conclusion: The high quality and quantity of current method, no enzymatic processing and accordingly its low cost, make it appropriate for DNA extraction not only from human but also from animal blood samples in any molecular biology labs.

  18. Towards a rapid molecular diagnostic for melioidosis: comparison of DNA extraction methods from clinical specimens

    Science.gov (United States)

    Richardson, Leisha J; Kaestli, Mirjam; Mayo, Mark; Bowers, Jolene R; Tuanyok, Apichai; Schupp, Jim; Engelthaler, David; Wagner, David M; Keim, Paul S; Currie, Bart J

    2011-01-01

    Optimising DNA extraction from clinical samples for Burkholderia pseudomallei Type III secretion system real-time PCR in suspected melioidosis patients confirmed that urine and sputum are useful diagnostic samples. Direct testing on blood remains problematic; testing DNA extracted from plasma was superior to DNA from whole blood or buffy coat. PMID:22108495

  19. Microbial DNA extraction from intestinal biopsies is improved by avoiding mechanical cell disruption

    Science.gov (United States)

    Carbonero, Franck; Nava, Gerardo M.; Benefiel, Ann C.; Greenberg, Eugene; Gaskins, H. Rex

    2011-01-01

    Currently, standard protocols for microbial DNA extraction from intestinal tissues do not exist. We assessed the efficiency of a commercial kit with and without mechanical disruption. Better quality DNA was obtained without mechanical disruption. Thus, it appears that bead-beating is not required for efficient microbial DNA extraction from intestinal biopsies. PMID:21820015

  20. Comparison of three methods of DNA extraction from human bones with different degrees of degradation.

    Science.gov (United States)

    Jakubowska, Joanna; Maciejewska, Agnieszka; Pawłowski, Ryszard

    2012-01-01

    There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol-chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol-chloroform protocols are more efficient for successful DNA extraction.

  1. Identification of multiple HPV types on spermatozoa from human sperm donors.

    Science.gov (United States)

    Kaspersen, Maja D; Larsen, Peter B; Ingerslev, Hans Jakob; Fedder, Jens; Petersen, Gert Bruun; Bonde, Jesper; Höllsberg, Per

    2011-03-29

    Human papillomaviruses (HPV) may cause sexually transmitted disease. High-risk types of HPV are involved in the development of cervical cell dysplasia, whereas low-risk types may cause genital condyloma. Despite the association between HPV and cancer, donor sperm need not be tested for HPV according to European regulations. Consequently, the potential health risk of HPV transmission by donor bank sperm has not been elucidated, nor is it known how HPV is associated with sperm. The presence of 35 types of HPV was examined on DNA from semen samples of 188 Danish sperm donors using a sensitive HPV array. To examine whether HPV was associated with the sperm, in situ hybridization were performed with HPV-6, HPV-16 and -18, and HPV-31-specific probes. The prevalence of HPV-positive sperm donors was 16.0% and in 66.7% of these individuals high-risk types of HPV were detected. In 5.3% of sperm donors, two or more HPV types were detected. Among all identified HPV types, 61.9% were high-risk types. In situ hybridization experiments identified HPV genomes particularly protruding from the equatorial segment and the tail of the sperm. Semen samples from more than one in seven healthy Danish donors contain HPV, most of them of high-risk types binding to the equatorial segment of the sperm cell. Most HPV-positive sperm showed decreased staining with DAPI, indicative of reduced content of DNA. Our data demonstrate that oncogenic HPV types are frequent in men.

  2. Construction and Identification of a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein

    Institute of Scientific and Technical Information of China (English)

    梅泉; 李双; 刘萍; 奚玲; 王世宣; 孟玉菡; 刘杰; 杨欣慰; 卢运萍; 汪辉

    2010-01-01

    By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...

  3. Construction and identification of PVAX1-HPV58mE6E7Fc-hIL12 composite DNA vaccine vector%人乳头瘤病毒58型治疗性复合DNA疫苗载体的构建

    Institute of Scientific and Technical Information of China (English)

    王鹤; 于继云; 李力

    2012-01-01

    目的:构建和表达人乳头瘤病毒(Human papilloma virus,HPV)58型相关宫颈癌治疗用 PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体.方法:将HPV58mE6E7融合基因片段插入真核表达载体pCI-Fc-GPI中,构建重组真核表达质粒pCI-sig-HPV58mE6E7-Fc-GPI.再将得到的sig-HPV58mE6E7-Fc-GPI片段插入新型疫苗载体PVAX1-IRES-hIL12中,构建PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体,流式细胞术、免疫荧光及ELISA分别检测该疫苗载体的表达.结果:经限制性内切酶鉴定及测序分析证实PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体构建成功.流式细胞术、免疫荧光及ELISA检测表明该疫苗载体中的融合抗原sig-HPV58mE6E7-Fc-GPI及分子佐剂人白介素12(hIL12)能够同时实现真核表达.结论:PVAX1-HPV58mE6E7Fc-hIL12复合DNA疫苗载体可作为治疗HPV58相关肿瘤及其癌前病变的候选疫苗载体.%Objective: To construct and express a composite DNA vaccine vector named PVAXl-HPV58mE6E7Fc-hIL12 for human papilomavirus 58(HPV58)-associated cervical cancer. Methods:HPV58mE6E7 fusion gene was inserted into pCI-Fc-GPI eu-karyotic expression vector, constructing a recombinant plasmid named pCl-sig-HPV58mE6E7-r c-GPI. The fragment of sig-HPV58mE6E7-Fc-GPI was inserted into the novel vaccine vector PVAX1-IRES-hIL12, constructing PVAXl-HPV58mE6E7Fc-hIL12 composite DNA vaccine vector. Flow cytometry, immunofluorescence and ELBA were used to detect expression of the vaccine. Re-sllltS;PVAXl-HPV58mE6E7Fc-hIL12 vaccine vector was successfully constructed and identified by restriction endonuclease and sequencing analysis. The expression analysis showed that fusion antigen sig-HPV58mE6E7-Fc-GPI and molecular adjuvant human inter-leukin 12(hIL12) can be realized at the same time. Conchlsion:PVAXl-HPV58mE6E7Fc-hIL12 can be taken as a candidate of thera-peutic composite DNA vaccine vector for HPV 58-associated. Tumors and. Tneir precancerous translormations.

  4. DNA Extraction by Isotachophoresis in a Microfluidic Channel

    Energy Technology Data Exchange (ETDEWEB)

    Stephenson, S J

    2011-08-10

    Biological assays have many applications. For example, forensics personnel and medical professionals use these tests to diagnose diseases and track their progression or identify pathogens and the host response to them. One limitation of these tests, however, is that most of them target only one piece of the sample - such as bacterial DNA - and other components (e.g. host genomic DNA) get in the way, even though they may be useful for different tests. To address this problem, it would be useful to extract several different substances from a complex biological sample - such as blood - in an inexpensive and efficient manner. This summer, I worked with Maxim Shusteff at Lawrence Livermore National Lab on the Rapid Automated Sample Prep project. The goal of the project is to solve the aforementioned problem by creating a system that uses a series of different extraction methods to extract cells, bacteria, and DNA from a complex biological sample. Biological assays can then be run on purified output samples. In this device, an operator could input a complex sample such as blood or saliva, and would receive separate outputs of cells, bacteria, viruses, and DNA. I had the opportunity to work this summer with isotachophoresis (ITP), a technique that can be used to extract nucleic acids from a sample. This technique is intended to be the last stage of the purification device. Isotachophoresis separates particles based on different electrophoretic mobilities. This technique is convenient for out application because free solution DNA mobility is approximately equal for DNA longer than 300 base pairs in length. The sample of interest - in our case DNA - is fed into the chip with streams of leading electrolyte (LE) and trailing electrolyte (TE). When an electric field is applied, the species migrate based on their electrophoretic mobilities. Because the ions in the leading electrolyte have a high electrophoretic mobility, they race ahead of the slower sample and trailing

  5. DNA, RNA, and Protein Extraction: The Past and The Present

    Directory of Open Access Journals (Sweden)

    Beow Chin Yiap

    2009-01-01

    Full Text Available Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.

  6. Extraction and DNA Digestion of 5'-Phosphodiesterase from Malt Root

    Institute of Scientific and Technical Information of China (English)

    ZOU Hechang; CAI Guangqi; CAI Wen; LI Hailong; GU Yi; PARK Yongdoo; MENG Fanguo

    2008-01-01

    This study investigated the extraction of 5'-phosphodiesterase from malt root and the degradation of nucleic acids by this enzyme.The extraction used grade precipitation with ammonium sulfate and enzy-matic hydrolysis.Samples were assayed using the modified Bradford method and high performance liquid chromatography.The results show that 5'-phosphodiesterase is isolated by grade precipitation with 30% and 80% saturation of ammonium sulfate and can be utilized to degrade deoxydbonucleic acid.The hydrolysate has four kinds of deoxynucleotides: 5'-dCMP,5'-dTMP,5'-dAMP,and 5'-dGMP.The optimum reaction tem-perature is 70℃,and the optimum pH is 5.5-6.0 for the reaction.The percentage of deoxynucleotides indi-ceted by the China Pharmacopoeia (2000 edition) in the product is over 70%.The extraction of 5'-phosphodiesterase from malt root is shown to be possible and economical.Products from the enzymatic hydrolysate of DNA meet the pharmacopoeia.

  7. 评价液基细胞学及HPV-DNA检测在宫颈癌筛查中的价值%Evaluation of the Value of Liquid based Cytology and HPV-DNA Test in the Screening of Cervical Cancer

    Institute of Scientific and Technical Information of China (English)

    彭幼香

    2016-01-01

    Objective To explore the value of liquid based cytology test and HPV-DNA test in the screening of cervical cancer. Methods Randomly selected in the Department of gynecology and obstetrics in our hospital in 2015 10 months to 2016 800 cases of during the month of may receive medical treatment as the research object of discussion, 800 cases of medical treatment were treated with detection of HPV-DNA and TCT, the two test results if one of them is abnormal, accept pathological examination, the diagnosis fault gold standard for pathological examination and calculated predictive value, positive predictive value, sensitivity and speciifcity of the detection of negative and index, and the results of the analysis. Results The difference between the area under the joint detection and ASC-H belongs to 800 cases of research object and HPV-DNA examination results showed positive in 104 cases, accounted for 13%;TCT inspection a total of 93 patients with abnormal proportion was 11.63%, including a total of 7 cases of ASC-US 52 cases of LSIL, a total of 13 cases, HSIL a total of 18 cases of SCC were 3 cases. A total of 227 patients who underwent pathological examination, examination results showed abnormal in 34 cases, SCC containing a total of 3 cases of CIN in 31 cases. The negative predictive value was higher.HPV-DNA TCT detection way of alone of the ROC curve area respectively 0.629 and 0.761 and the two kinds of detection methods, curve 0.902, and separate use of HPV-DNA and TCT detection methods were compared, the group was statistically signiifcant (P<0.05). Conclusion The combined use of HPV-DNA and TCT two kinds of detection methods, in the cervical cancer screening for high accuracy, is a more ideal way of screening.%目的:探讨分析液基细胞学及HPV-DNA检测在宫颈癌筛查中的价值。方法随机选取我院妇产科2015年10月到2016年4月期间所接受的体检就诊者800例作为本研究讨论对象,800例体检就诊者均接受HPV-DNA和TCT

  8. Commercially available molecular tests for human papillomaviruses (HPV): 2015 update.

    Science.gov (United States)

    Poljak, Mario; Kocjan, Boštjan J; Oštrbenk, Anja; Seme, Katja

    2016-03-01

    Commercial molecular tests for human papillomaviruses (HPV) are invaluable diagnostic tools in cervical carcinoma screening and management of women with cervical precancerous lesions as well as important research tools for epidemiological studies, vaccine development, and implementation and monitoring of vaccination programs. In this third inventory of commercial HPV tests, we identified 193 distinct commercial HPV tests and at least 127 test variants available on the market in 2015, which represents a 54% and 79% increase in the number of distinct HPV tests and variants, respectively, in comparison to our last inventory performed in 2012. Identified HPV tests were provisionally divided into eight main groups and several subgroups. Among the 193 commercial HPV tests, all but two target alpha-HPV types only. Although the number of commercial HPV tests with at least one published study in peer-reviewed literature has increased significantly in the last three years, several published performance evaluations are still not in line with agreed-upon standards in the HPV community. Manufacturers should invest greater effort into evaluating their products and publishing validation/evaluation results in peer-reviewed journals. To achieve this, more clinically oriented external quality-control panels and initiatives are required. For evaluating the analytical performance of the entire range of HPV tests currently on the market, more diverse and reliable external quality-control programs based on international standards for all important HPV types are indispensable. The performance of a wider range of HPV tests must be promptly evaluated on a variety of alternative clinical specimens. In addition, more complete HPV assays containing validated sample-extraction protocols and appropriate internal controls are urgently needed. Provision of a broader range of automated systems allowing large-scale HPV testing as well as the development of reliable, rapid, and affordable molecular

  9. Racial differences in the incidence and clearance of human papillomavirus (HPV): The HPV in Men (HIM) Study

    Science.gov (United States)

    Schabath, Matthew B.; Villa, Luisa L.; Lin, Hui-Yi; Fulp, William J.; Akogbe, Gabriel O.; Abrahamsen, Martha E.; Papenfuss, Mary R.; Lazcano-Ponce, Eduardo; Salmerón, Jorge; Quiterio, Manuel; Giuliano, Anna R.

    2013-01-01

    Background This analysis assessed the acquisition (incidence) and persistence (clearance) of HPV infection by self-reported race among men in The HPV in Men (HIM) Study, a multinational prospective study of the natural history of genital HPV infections. Methods Self-reported race was categorized as White, Black, Asian/Pacific Islander (PI), or multiple and mixed race. Genital samples were combined for HPV DNA testing and categorized by any-, oncogenic-, and non-oncogenic HPV infections. Results Asian/PI race had significantly the lowest incidence of any-, oncogenic-, and non-oncogenic HPV infection (P < 0.001). In multivariable analyses Asian/PI race was associated with a lower probability of acquiring any- (HR=0.63; 95% CI 0.42–0.95) and non-oncogenic HPV infection (HR=0.61; 95% CI 0.40–0.93) when compared to Whites. No significant associations were evident for Asian/PI race for clearance. Multiple and mixed race was significantly associated with lower probability of acquiring non-oncogenic HPV infection (HR=0.83; 95% CI 0.69–0.99) and borderline significant associations were observed for any HPV (HR=0.91) and oncogenic infections (HR=0.92). Multiple and mixed race was associated with a lower probability of clearing any- (HR=0.92; 95% CI 0.84–1.00) and oncogenic HPV infections (HR=0.85; 95% CI 0.75–0.95). Conclusion Asian/PI race had the lowest incidence of HPV and exhibited a lower probability of acquiring new HPV infections. Multiple and mixed race had the second lowest incidence of infection and was associated with a lower probability of acquiring and clearing a HPV infection. Impact Race-specific differences in HPV infection could be due to behavior, innate genetic differences, or circulating intratypic HPV variants. PMID:23872745

  10. Silica-Based Solid Phase Extraction of DNA on a Microchip

    Institute of Scientific and Technical Information of China (English)

    陈晓芳; 沈科跃; 刘鹏; 郭旻; 程京; 周玉祥

    2004-01-01

    Micro total analysis systems for chemical and biological analysis have attracted much attention.However,microchips for sample preparation and especially DNA purification are still underdeveloped.This work describes a solid phase extraction chip for purifying DNA from biological samples based on the adsorption of DNA on bare silica beads prepacked in a microchannel.The chip was fabricated with poly-dimethylsiloxane.The silica beads were packed in the channel on the chip with a tapered microchannel to form the packed bed.Fluorescence detection was used to evaluate the DNA adsorbing efficiency of the solid phase.The polymerase chain reaction was used to evaluate the quality of the purified DNA for further use.The extraction efficiency for the DNA extraction chip is approximately 50% with a 150-nL extraction volume.Successful amplification of DNA extracted from human whole blood indicates that this method is compatible with the polymerase chain reaction.

  11. Touchdown General Primer (GP5+/GP6+ PCR and optimized sample DNA concentration support the sensitive detection of human papillomavirus

    Directory of Open Access Journals (Sweden)

    Simmons-Arnold Linda

    2005-11-01

    Full Text Available Abstract Background The GP5+/GP6+ PCR assay is a well-established HPV detection technique. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. Methods Firstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng–100 ng diluted in a background of C-33A DNA (100 ng-2 μg. Secondly, the detection of small quantities (15ag-1.5pg of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56 diluted in C-33A DNA was investigated. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors were tested for HPV. Six different PCR protocols were assessed. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. Results HPV detection sensitivity was dependent on the total amount of DNA in a PCR. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 μg or 1 μg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (~28 copies HPV-16 in 500 ng or 100 ng background DNA. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 μg or 1 μg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. HPV recombinant plasmids were each detected with high (albeit varying sensitivity by a touchdown protocol. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA than by a touchdown approach (15fg detection limit. HPV-52 was not amplified by the standard protocol at the dilutions tested. Seventeen different HPV types were demonstrated in 47/65 (72% abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. Twenty-one different HPV types were recorded in 111/114 (97% vaginal lesions. Multiple infections were also detectable using a touchdown

  12. Evaluation of DNA Extraction Methods Suitable for PCR-based Detection and Genotyping of Clostridium botulinum

    DEFF Research Database (Denmark)

    Auricchio, Bruna; Anniballi, Fabrizio; Fiore, Alfonsina

    2013-01-01

    Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient...... in terms of cost, time, labor, and supplies. Eleven botulinum toxin–producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin–producing clostridia were used to compare 4 DNA extraction procedures: Chelex® 100 matrix, Phenol......-Cloroform-Isoamyl alcohol, NucliSENS® magnetic extraction kit, and DNeasy® Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy® Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However...

  13. Comparison of different protocols for the extraction of microbial DNA from reef corals

    Directory of Open Access Journals (Sweden)

    H.F. Santos

    2012-06-01

    Full Text Available This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio. Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

  14. Comparison of different protocols for the extraction of microbial DNA from reef corals

    Science.gov (United States)

    Santos, H.F.; Carmo, F.L.; Leite, D.C.A.; Jesus, H.E.; Maalouf, P. De Carvalho; Almeida, C.; Soriano, A.U.; Altomari, D.; Suhett, L.; Vólaro, V.; Valoni, E.; Francisco, M.; Vieira, J.; Rocha, R.; Sardinha, B.L.; Mendes, L.B.; João, R.R.; Lacava, B.; Jesus, R.F.; Sebastian, G.V.; Pessoa, A.; van Elsas, J.D.; Rezende, R.P.; Pires, D.O.; Duarte, G.; Castro, C.B.; Rosado, A.S.; Peixoto, R.S.

    2012-01-01

    This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals. PMID:24031859

  15. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below

  16. Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction.

    Science.gov (United States)

    Jackson, D P; Lewis, F A; Taylor, G R; Boylston, A W; Quirke, P

    1990-06-01

    Several DNA extraction techniques were quantitatively and qualitatively compared using both fresh and paraffin wax embedded tissue and their suitability investigated for providing DNA and RNA for the polymerase chain reaction (PCR). A one hour incubation with proteinase K was the most efficient DNA extraction procedure for fresh tissue. For paraffin wax embedded tissue a five day incubation with proteinase K was required to produce good yields of DNA. Incubation with sodium dodecyl sulphate produced very poor yields, while boiling produced 20% as much DNA as long enzyme digestion. DNA extracted by these methods was suitable for the PCR amplification of a single copy gene. Proteinase K digestion also produced considerable amounts of RNA which has previously been shown to be suitable for PCR analysis. A delay before fixation had no effect on the amount of DNA obtained while fixation in Carnoy's reagent results in a much better preservation of DNA than formalin fixation, allowing greater yields to be extracted.

  17. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    2011-01-01

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below simila

  18. DNA is a co-factor for its own replication in Xenopus egg extracts

    NARCIS (Netherlands)

    Lebofsky, Ronald; van Oijen, Antoine M.; Walter, Johannes C.

    2011-01-01

    Soluble Xenopus egg extracts efficiently replicate added plasmids using a physiological mechanism, and thus represent a powerful system to understand vertebrate DNA replication. Surprisingly, DNA replication in this system is highly sensitive to plasmid concentration, being undetectable below simila

  19. Cancer Immunology and HPV.

    Science.gov (United States)

    Wollenberg, Barbara

    HNSCC is a heterogeneous group of tumors located in the oral cavity, oropharynx, hypopharynx and larynx. Originally, tobacco and alcohol exposures were the main risk factors for HNSCC. In the last two decades, HPV infections have been identified as a risk factor for HNSCC, especially for oropharyngeal tumors. Whereas the HPV-induced oropharyngeal carcinomas predominantly express the HPV16 related E6 and E7 oncoproteins, the HPV-negative HNSCC are associated with an overexpression of p53. However, if the therapy successes for HPV-negative and HPV-positive HNSCCs are compared, there are significantly higher total survival rates for HPV-positive oropharyngeal tumors compared to HPV-negative tumors. It is important to understand this phenomenon in order to improve and adapt therapy concepts.

  20. Prevalence of HPV and EBV infection and their relationship with the p53 and PCNA expression in oral carcinoma patients.

    Directory of Open Access Journals (Sweden)

    Dayahindara Veitía

    2017-04-01

    Full Text Available Introduction: Infection caused by potentially oncogenic viruses, such as HPV and EBV, favors the role of certain oncoproteins that can induce dysplasias and malignant lesions. Objective: To evaluate the prevalence of HPV and EBV and their relation with the expression of p53 and PCNA in patients with oral carcinoma. Methodology: Twenty-seven oral squamous cell carcinomas (OSCC were evaluated; DNA extraction was conducted using the QIAamp DNA mini kit; viral detection was obtained using the INNO-LiPA kit for HPV, and nested PCR was used for EBV. The evaluation of molecular markers was performed through immunohistochemical staining. Results: The mean age of the patients was 60.55±13.94 years, and 52% of these were female. Of the patients, 59% were tobacco users and 63% were alcohol consumers. HPV was detected in 70% of the patients with the predominance of genotype 16 (60%. As for EBV infection, it was observed in 59% of cases. p53 and PCNA immunopositivity corresponded to 44% and 59%, respectively. The tongue was the anatomical location with highest positivity for both viruses as well as for the expression of molecular markers. The 48% of the cases presented infection by both viruses. Conclusion: HPV and EBV infection together with the expression of p53 and PCNA were more frequently observed in advanced stages of the disease, suggesting a more relevant role in the progression than in tumor genesis.

  1. DNA extraction of birch leaves by improved CTAB method and optimization of its ISSR system

    Institute of Scientific and Technical Information of China (English)

    PAN hua; YANG Chuan-ping; WEI Zhi-gang; JIANG Jing

    2006-01-01

    The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable not only for genomic DNA extraction of birch but also for that of other plants. The purity of genomic DNA extracted by the multi-times STE-CTAB extraction method is higher than that by one time STE-CTAB method, and it does not need the process of RNase. The factors of influencing ISSR system were explored based on the genomic DNA of birch extracted by the two methods. The optimal conditions for ISSR system were determined as follows: cycles of denaturation for 30 s at 94℃, annealing for 30 s at 51 ℃, extension for 30 s at 72℃, and a final 7 min extension at 72 ℃.

  2. DNA extract characterization process for microbial detection methods development and validation

    OpenAIRE

    2012-01-01

    Background Quantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods. Results D...

  3. Comparison of DNA quantification methodology used in the DNA extraction protocol for the UK Biobank cohort.

    Science.gov (United States)

    Welsh, Samantha; Peakman, Tim; Sheard, Simon; Almond, Rachael

    2017-01-05

    UK Biobank is a large prospective cohort study in the UK established by the Medical Research Council (MRC) and the Wellcome Trust to enable approved researchers to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. A wide range of phenotypic data has been collected at recruitment and has recently been enhanced by the UK Biobank Genotyping Project. All UK Biobank participants (500,000) have been genotyped on either the UK Biobank Axiom® Array or the Affymetrix UK BiLEVE Axiom® Array and the workflow for preparing samples for genotyping is described. The genetic data is hoped to provide further insight into the genetics of disease. All data, including the genetic data, is available for access to approved researchers. Data for two methods of DNA quantification (ultraviolet-visible spectroscopy [UV/Vis]) measured on the Trinean DropSense™ 96 and PicoGreen®) were compared by two laboratories (UK Biobank and Affymetrix). The sample processing workflow established at UK Biobank, for genotyping on the custom Affymetrix Axiom® array, resulted in high quality DNA (average DNA concentration 38.13 ng/μL, average 260/280 absorbance 1.91). The DNA generated high quality genotype data (average call rate 99.48% and pass rate 99.45%). The DNA concentration measured on the Trinean DropSense™ 96 at UK Biobank correlated well with DNA concentration measured by PicoGreen® at Affymetrix (r = 0.85). The UK Biobank Genotyping Project demonstrated that the high throughput DNA extraction protocol described generates high quality DNA suitable for genotyping on the Affymetrix Axiom array. The correlation between DNA concentration derived from UV/Vis and PicoGreen® quantification methods suggests, in large-scale genetic studies involving two laboratories, it may be possible to remove the DNA quantification step in one laboratory without affecting downstream analyses. This would result in

  4. Analysis of E2 gene integrity in HPV16 and HPV58 viruses isolated from women with cervical pathology

    Science.gov (United States)

    González-Losa, María del R; Puerto-Solis, Marylin; Tenorio Ruiz, Juan; Rosado-López, Ariel I; Hau-Aviles, Oscar; Ayora-Talavera, Guadalupe; Cisneros-Cutz, Isidro; Conde-Ferráez, Laura

    2016-01-01

    Integration of human papillomavirus (HPV) DNA into human cells accompanied by the disruption of the viral genome has been described as a prerequisite for cancer development. This study aimed to investigate E2 gene integrity of HPV16 and HPV58 viruses isolated from infected women with cervical lesions. Forty-two HPV16- and 31 HPV58-positive samples were analysed. E2 integrity was assumed when all fragments covering the E2 gene were amplified with specific polymerase chain reaction primers. Overall, in 59% of the samples, at least one fragment was not amplified in HPV16- (57%) and HPV58-positive samples (61%). Samples from high-grade squamous intraepithelial lesions had the highest frequency of E2 gene disruptions (73%), followed by samples from low-grade squamous intraepithelial lesions (63%) and, finally, samples from invasive cervical cancer (35%). Association between the integrity status of the E2 gene, and lesion grade was assessed by the chi-squared test applied to the combined set of viruses (p = 0.6555) or to populations of the same virus type (HPV58, p = 0.3101; HPV16, p = 0.3024). In conclusion, in this study, no association was found between the presence of E2 gene disruptions and the grade of cervical lesions caused by HPV16 and HPV58. PMID:27812600

  5. TMEM16A/ANO1 is differentially expressed in HPV-negative versus HPV-positive head and neck squamous cell carcinoma through promoter methylation.

    Science.gov (United States)

    Dixit, Ronak; Kemp, Carolyn; Kulich, Scott; Seethala, Raja; Chiosea, Simion; Ling, Shizhang; Ha, Patrick K; Duvvuri, Umamaheswar

    2015-11-13

    Head and neck squamous cell carcinoma (HNSCC) has a variety of causes. Recently, the human papilloma virus (HPV) has been implicated in the rising incidence of oropharyngeal cancer and has led to variety of studies exploring the differences between HPV-positive and HPV-negative HNSCC. The calcium-activated chloride channel TMEM16A is overexpressed in a variety of cancers, including HNSCC, but whether or not it plays different roles in HPV-positive and HPV-negative HNSCC is unknown. Here, we demonstrate that TMEM16A is preferentially overexpressed in HPV-negative HNSCC and that this overexpression of TMEM16A is associated with decreased patient survival. We also show that TMEM16A expression is decreased in HPV-positive HNSCC at the DNA, RNA, and protein levels in patient samples as well as cell lines. We demonstrate that the lower levels of TMEM16A expression in HPV-positive tumors can be attributed to both a combination of copy number alteration and promoter methylation at the DNA level. Additionally, our cellular data show that HPV-negative cell lines are more dependent on TMEM16A for survival than HPV-positive cell lines. Therefore, we suspect that the down-regulation of TMEM16A in HPV-positive HNSCC makes TMEM16A a poor therapeutic target in HPV-positive HNSCC, but a potentially useful target in HPV-negative HNSCC.

  6. An improved method with a wider applicability to isolate plant mitochondria for mtDNA extraction

    OpenAIRE

    2015-01-01

    Background Mitochondria perform a principal role in eukaryotic cells. Mutations in mtDNA can cause mitochondrial dysfunction and are frequently associated with various abnormalities during plant development. Extraction of plant mitochondria and mtDNA is the basic requirement for the characterization of mtDNA mutations and other molecular studies. However, currently available methods for mitochondria isolation are either tissue specific or species specific. Extracted mtDNA may contain substant...

  7. The clinical implementation of primary HPV screening.

    Science.gov (United States)

    Mariani, Luciano; Igidbashian, Sarah; Sandri, Maria Teresa; Vici, Patrizia; Landoni, Fabio

    2017-03-01

    To evaluate, from a gynecology perspective, the transition from cytology-based HPV screening to primary HPV screening. Studies examining switching from cytology-based screening to primary HPV-DNA testing with triaging of patients with positive test results were retrieved and reviewed, with a particular focus on screening in an Italian setting. The increased complexity of patient-management decisions when implementing HPV-based screening was a critical issue discussed in the literature. The change in strategy represents a paradigm shift in moving from a medical perspective of identifying the disease in individual patients, to a public-healthcare perspective of excluding HPV from the healthy population and identifying a small sub-group of individuals at increased risk of HPV. With knowledge about HPV screening evolving rapidly, new programs and related algorithms need to be sufficiently flexible to be adjusted according to ongoing research and the validation of new assays. The establishment of a national working group (including epidemiologists, gynecologists, pathologists, and healthcare providers) will be necessary to properly implement and govern this important technical and cultural transition. © 2016 International Federation of Gynecology and Obstetrics.

  8. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants

    Directory of Open Access Journals (Sweden)

    Yang Moon-Sik

    2004-09-01

    Full Text Available Abstract Background DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. Results We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1. After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. Conclusion This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

  9. Rapid and reliable extraction of genomic DNA from various wild-type and transgenic plants.

    Science.gov (United States)

    Kang, Tae-Jin; Yang, Moon-Sik

    2004-09-02

    DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants. We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes. This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction.

  10. Decision-analytic modeling to evaluate the long-term effectiveness and cost-effectiveness of HPV-DNA testing in primary cervical cancer screening in Germany

    Directory of Open Access Journals (Sweden)

    Krämer, Alexander

    2010-01-01

    Full Text Available Background: Persistent infections with high-risk types of human papillomavirus (HPV are associated with the development of cervical neoplasia. Compared to cytology HPV testing is more sensitive in detecting high-grade cervical cancer precursors, but with lower specificity. HPV based primary screening for cervical cancer is currently discussed in Germany. Decisions should be based on a systematic evaluation of the long-term effectiveness and cost-effectiveness of HPV based primary screening. Research questions: What is the long-term clinical effectiveness (reduction in lifetime risk of cervical cancer and death due to cervical cancer, life years gained of HPV testing and what is the cost-effectiveness in Euro per life year gained (LYG of including HPV testing in primary cervical cancer screening in the German health care context? How can the screening program be improved with respect to test combination, age at start and end of screening and screening interval and which recommendations should be made for the German health care context? Methods: A previously published and validated decision-analytic model for the German health care context was extended and adapted to the natural history of HPV infection and cervical cancer in order to evaluate different screening strategies that differ by screening interval, and tests, including cytology alone, HPV testing alone or in combination with cytology, and HPV testing with cytology triage for HPV-positive women. German clinical, epidemiological and economic data were used. In the absence of individual data, screening adherence was modelled independently from screening history. Test accuracy data were retrieved from international meta-analyses. Predicted outcomes included reduction in lifetime-risk for cervical cancer cases and deaths, life expectancy, lifetime costs, and discounted incremental cost-effectiveness ratios (ICER. The perspective of the third party payer and 3% annual discount rate were

  11. Comparison of commercial kits for the extraction of DNA from paddy soils.

    Science.gov (United States)

    Knauth, S; Schmidt, H; Tippkötter, R

    2013-03-01

    The objective of this study was to compare the extraction efficiency of commercial DNA kits by evaluating the quantity and purity of DNA extracts obtained from paddy soils. DNA was extracted from three paddy soils using the FastDNA® SPIN kit for soil (FD), the innuSPEED soil DNA kit (INS) and the NucleoSpin® soil kit (NSP). DNA extracts were analysed by agarose gel electrophoresis and UV spectroscopy. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses were conducted to evaluate the potential bias of the DNA extractions on fingerprinting techniques. Regarding the quantity and the purity of the extracted DNA, the NSP kit was detected superior to the FD kit, while the INS kit failed to extract detectable amounts of DNA. DGGE fingerprints generated from PCR products (FD, NSP) showed high levels of similarity for the amplified 16S rRNA genes of methanogenic archaea (>95%) and bacteria (up to 100%) in each soil. This study suggested that the recently introduced NSP kit allowed for the adjustment of the lysis buffer composition to the soil of interest and is at least equivalent to the well-established FD kit for the extraction of DNA from paddy soils. The choice of commercial kits (FD, INS, NSP) has been of great importance regarding the quantity and purity of DNA extracted from paddy soils in this study. The composition of the cell lysis buffer represented a key component for successful extractions of DNA from different soils. The possibility of adjusting the lysis buffer to the soil of interest as well as the reproducibility of DGGE banding patterns makes the recently introduced NSP kit a strong competitor to the well-established FD kit for the extraction of DNA from paddy soils. © 2012 The Society for Applied Microbiology.

  12. DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

    Science.gov (United States)

    Moncada, Ximena; Payacán, Claudia; Arriaza, Francisco; Lobos, Sergio; Seelenfreund, Daniela; Seelenfreund, Andrea

    2013-01-01

    Background Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. Methodology We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. Conclusions Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials. PMID:23437166

  13. DNA extraction and amplification from contemporary Polynesian bark-cloth.

    Directory of Open Access Journals (Sweden)

    Ximena Moncada

    Full Text Available BACKGROUND: Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. METHODOLOGY: We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. CONCLUSIONS: Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials.

  14. [Comparing and evaluating six methods of extracting human genomic DNA from whole blood].

    Science.gov (United States)

    Chang, Jing-Jing; Zhang, Su-Hua; Li, Li

    2009-04-01

    Comparing the differences in purity and yield among six methods of extracting human genomic DNA from whole blood, which included Classic Phenol-chloroform extraction, modified combined technique composed of improved Phenol-chloroform extraction and Chelex-100 extraction, Chelex-100 extraction, IQ, Qiagen and SP. Ten samples of intravenous whole blood (5 mL/sample) were collected and human genomic DNA was extracted with these six methods. The purity and concentration of the DNA products were detected by ultraviolet spectrophotometry and fluorescent quantitation technique, the yield was calculated and tested with statistical software. The Chelex-100 extraction was inferior in DNA purity to other methods while the other five methods showed no statistical difference. Modified combined technique was the poorest and IQ was the best in yield among the six methods of extraction. Statistical result showed that the extraction with high quality kits was better than that with classic Phenol-chloroform extraction, Chelex-100 extraction and modified combined technique composed of improved Phenol-chloroform. There was statistical difference between them. Comparing to Phenol-chloroform extraction and Chelex-100 extraction, high quality kits are more useful in DNA extraction from forensic materials.

  15. Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Ramos, W; Tappe, N; Talamantez, J; Friedberg, E C; Tomkinson, A E

    1997-01-01

    Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joi...

  16. A kinetic analysis of strand breaks on large DNA induced by cigarette smoke extract

    Science.gov (United States)

    Kurita, Hirofumi; Takata, Tatsuya; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2010-06-01

    We report a kinetic analysis of strand breakages on large DNA molecules induced by cigarette smoke extract (CSE), an extract of soluble cigarette smoke components. Previously, this DNA damage was analyzed by agarose gel electrophoresis, whereas we used fluorescence to kinetically analyze damage to individual DNA molecules. CSE caused a marked change in length of DNA molecules. The rate of CSE-induced double-strand breakage on large random-coiled DNA molecules was determined using a simple theoretical model, allowing the facile estimation of the rate of double-strand breaks on large DNA molecules.

  17. [Research advances on DNA extraction methods from peripheral blood mononuclear cells].

    Science.gov (United States)

    Wang, Xiao-Ying; Yu, Chen-Xi

    2014-10-01

    DNA extraction is a basic technology of molecular biology. The purity and the integrality of DNA structure are necessary for different experiments of gene engineering. As commonly used materials in the clinical detection, the fast, efficient isolation and extraction of genomic DNA from peripheral blood mononuclear cells is very important for the inspection and analysis of clinical blood. At present, there are many methods for extracting DNA, such as phenol-chloroform method, salting out method, centrifugal adsorption column chromatography method (artificial methods), magnetic beads (semi-automatic method) and DNA extraction kit. In this article, a brief review of the principle for existing DNA blood extraction method, the specific steps and the assessment of the specific methods briefly are summarized.

  18. Streamlining DNA barcoding protocols: automated DNA extraction and a new cox1 primer in arachnid systematics.

    Directory of Open Access Journals (Sweden)

    Nina Vidergar

    Full Text Available BACKGROUND: DNA barcoding is a popular tool in taxonomic and phylogenetic studies, but for most animal lineages protocols for obtaining the barcoding sequences--mitochondrial cytochrome C oxidase subunit I (cox1 AKA CO1--are not standardized. Our aim was to explore an optimal strategy for arachnids, focusing on the species-richest lineage, spiders by (1 improving an automated DNA extraction protocol, (2 testing the performance of commonly used primer combinations, and (3 developing a new cox1 primer suitable for more efficient alignment and phylogenetic analyses. METHODOLOGY: We used exemplars of 15 species from all major spider clades, processed a range of spider tissues of varying size and quality, optimized genomic DNA extraction using the MagMAX Express magnetic particle processor-an automated high throughput DNA extraction system-and tested cox1 amplification protocols emphasizing the standard barcoding region using ten routinely employed primer pairs. RESULTS: The best results were obtained with the commonly used Folmer primers (LCO1490/HCO2198 that capture the standard barcode region, and with the C1-J-2183/C1-N-2776 primer pair that amplifies its extension. However, C1-J-2183 is designed too close to HCO2198 for well-interpreted, continuous sequence data, and in practice the resulting sequences from the two primer pairs rarely overlap. We therefore designed a new forward primer C1-J-2123 60 base pairs upstream of the C1-J-2183 binding site. The success rate of this new primer (93% matched that of C1-J-2183. CONCLUSIONS: The use of C1-J-2123 allows full, indel-free overlap of sequences obtained with the standard Folmer primers and with C1-J-2123 primer pair. Our preliminary tests suggest that in addition to spiders, C1-J-2123 will also perform in other arachnids and several other invertebrates. We provide optimal PCR protocols for these primer sets, and recommend using them for systematic efforts beyond DNA barcoding.

  19. Innovative Graphite Oxide-Cellulose Based Material Specific for Genomic DNA Extraction

    Science.gov (United States)

    Akceoglu, Garbis Atam; Li, Oi Lun; Saito, Nagahiro

    2015-11-01

    Extraction of genomic DNA from various types of samples is often challenging for commercial silica spin column. In this study, we proposed graphite oxide (GO)/cellulose composite as an alternative material for genomic DNA extraction. The purity of DNA and extraction efficiency were compared to that of commercial silica product. In this study, the total weight % of GO was fixed at 4.15% in GO/Cellulose composite. Chewed gum, nail clip, cigarette bud paper, animal tissue and hair sample were used as various genomic DNA sources for extraction experiments. Among all types of samples, the extraction efficiencies were 4 to 12 times higher than that of commercial silica spin column. The absorbance ratio of 260 nm to 280 nm (A260/A280) of all samples ranged between 1.6 and 2.0. The results demonstrated that GO/Cellulose composites might serve as an innovative solid support material for genomic DNA extraction.

  20. DNA extraction from fresh-frozen and formalin-fixed, paraffin-embedded human brain tissue.

    Science.gov (United States)

    Wang, Jian-Hua; Gouda-Vossos, Amany; Dzamko, Nicolas; Halliday, Glenda; Huang, Yue

    2013-10-01

    Both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) human brain tissues are invaluable resources for molecular genetic studies of central nervous system diseases, especially neurodegenerative disorders. To identify the optimal method for DNA extraction from human brain tissue, we compared methods on differently-processed tissues. Fragments of LRRK2 and MAPT (257 bp and 483 bp/245 bp) were amplified for evaluation. We found that for FFPE samples, the success rate of DNA extraction was greater when using a commercial kit than a laboratory-based method (successful DNA extraction from 76% versus 33% of samples). PCR amplicon size and storage period were key factors influencing the success rate of DNA extraction from FFPE samples. In the fresh-frozen samples, the DNA extraction success rate was 100% using either a commercial kit (QIAamp DNA Micro) or a laboratory-based method (sample boiling in 0.1 mol/L NaOH, followed by proteinase K digestion, and then DNA extraction using Chelex-100) regardless of PCR amplicon length or tissue storage time. Although the present results demonstrate that PCR-amplifiable genomic DNA can be extracted from both fresh-frozen and FFPE samples, fresh brain tissue is recommended for DNA extraction in future neuropathological studies.

  1. THE HPV STATUS IN BLADDER CANCER, TUMOR MORPHOLOGICAL CHARACTERISTICS, AND CLINICAL FEATURES OF THE DISEASE

    Directory of Open Access Journals (Sweden)

    D. A. Golovina

    2014-01-01

    Full Text Available The data of medical records of 101 patients with urothelial bladder cancer (BC were compared with the results of laboratory detection of human papillomaviruses (HPV in the tumor tissue samples taken from these patients during transurethral resection. DNA of HPV 16, the major type of the virus responsible for the occurrence of cervical cancer, was previously detected in 38 samples; and oncogenes E6 and E7 mRNA and HPV 16 E7 oncoprotein were found in 13 of these samples. Comparison of HPV-positive and HPV-negative groups revealed that HPV-positive BC showed higher cell anaplasia than HPV-negative one; moreover, primary cancer was HPV-positive more frequently than recurrent cancer. Sex, age, muscular layer invasion did not correlate with the HPV positivity of BC. 

  2. THE HPV STATUS IN BLADDER CANCER, TUMOR MORPHOLOGICAL CHARACTERISTICS, AND CLINICAL FEATURES OF THE DISEASE

    Directory of Open Access Journals (Sweden)

    D. A. Golovina

    2014-07-01

    Full Text Available The data of medical records of 101 patients with urothelial bladder cancer (BC were compared with the results of laboratory detection of human papillomaviruses (HPV in the tumor tissue samples taken from these patients during transurethral resection. DNA of HPV 16, the major type of the virus responsible for the occurrence of cervical cancer, was previously detected in 38 samples; and oncogenes E6 and E7 mRNA and HPV 16 E7 oncoprotein were found in 13 of these samples. Comparison of HPV-positive and HPV-negative groups revealed that HPV-positive BC showed higher cell anaplasia than HPV-negative one; moreover, primary cancer was HPV-positive more frequently than recurrent cancer. Sex, age, muscular layer invasion did not correlate with the HPV positivity of BC. 

  3. A 9-valent HPV vaccine against infection and intraepithelial neoplasia in women

    DEFF Research Database (Denmark)

    Joura, Elmar A; Giuliano, Anna R; Iversen, Ole-Erik;

    2015-01-01

    BACKGROUND: The investigational 9-valent viruslike particle vaccine against human papillomavirus (HPV) includes the HPV types in the quadrivalent HPV (qHPV) vaccine (6, 11, 16, and 18) and five additional oncogenic types (31, 33, 45, 52, and 58). Here we present the results of a study...... of the efficacy and immunogenicity of the 9vHPV vaccine in women 16 to 26 years of age. METHODS: We performed a randomized, international, double-blind, phase 2b-3 study of the 9vHPV vaccine in 14,215 women. Participants received the 9vHPV vaccine or the qHPV vaccine in a series of three intramuscular injections...... on day 1 and at months 2 and 6. Serum was collected for analysis of antibody responses. Swabs of labial, vulvar, perineal, perianal, endocervical, and ectocervical tissue were obtained and used for HPV DNA testing, and liquid-based cytologic testing (Papanicolaou testing) was performed regularly. Tissue...

  4. Estimation of the overall burden of cancers, precancerous lesions, and genital warts attributable to 9-valent HPV vaccine types in women and men in Europe.

    Science.gov (United States)

    Hartwig, Susanne; St Guily, Jean Lacau; Dominiak-Felden, Géraldine; Alemany, Laia; de Sanjosé, Silvia

    2017-01-01

    In addition to cervical cancer, human papillomavirus (HPV) is responsible for a significant proportion of cancers and precancerous lesions of the vulva, vagina, anus, penis, head and neck, as well as genital warts. We estimated the annual number of new cases of these diseases attributable to 9-valent HPV vaccine types in women and men in Europe. The annual number of new cancers of the cervix, vulva, vagina, anus, penis, and selected head and neck sites in the population of the European Medicines Agency territory was estimated based on age-specific incidence rates extracted from Cancer Incidence in 5 Continents, Volume X and Eurostat population data for 2015. The annual number of new cancers attributable to 9-valent HPV vaccine types was estimated by applying the HPV attributable fraction from reference publications based on a large European multicenter study. For non-cervical cancers, HPV attributable fractions were based on oncogenically-active HPV infections only (i.e., detection of HPV DNA and either mRNA and/or p16 positivity). For precancerous lesions of the cervix, vulva, vagina, and anus, and for genital warts, previously published estimations were updated for the 2015 population. The annual number of new cancers attributable to 9-valent HPV vaccine types was estimated at 47,992 (95% bound: 39,785-58,511). Cervical cancer showed the highest burden (31,130 cases), followed by head and neck cancer (6,786 cases), anal cancer (6,137 cases), vulvar cancer (1,466 cases), vaginal cancer (1,360 cases), and penile cancer (1,113 cases). About 81% were estimated to occur in women and 19% in men. The annual number of new precancerous lesions (CIN2+, VIN2/3, VaIN2/3, and AIN2/3) and genital warts attributable to 9-valent HPV vaccine types was estimated at 232,103 to 442,347 and 680,344 to 844,391, respectively. The burden of cancers associated with 9-valent HPV vaccine types in Europe is substantial in both sexes. Head and neck cancers constitute a heavy burden

  5. Comparison of the immunogenicity of Cervarix® and Gardasil® human papillomavirus vaccines for oncogenic non-vaccine serotypes HPV-31, HPV-33, and HPV-45 in HIV-infected adults

    DEFF Research Database (Denmark)

    Nielsen, Lars Toft; Tolstrup, Martin; Müller, Martin;

    2014-01-01

    Individuals infected with human immunodeficiency virus (HIV) have excess risk of developing human papillomavirus (HPV)-related disease. A substantial fraction of HPV-associated cancers is caused by HPV serotypes not included in the currently available vaccines. Among healthy women, both Cervarix......(®) (HPV-16/18, GlaxoSmithKline Biologicals, GSK) and Gardasil(®) (HPV-6/11/16/18, Merck) have demonstrated partial cross-protection against certain oncogenic non-vaccine HPV-types. Currently, there are no available data on vaccine-induced cross-protection in men and little is known about cross......-reactive immunity after HPV-vaccination of HIV-infected individuals. In an investigator-initiated trial, we randomized 91 HIV-positive men and women to receive vaccination with Cervarix(®) or Gardasil(®). The HPV-DNA status of the participants was determined with pcr before and after immunization. Cross...

  6. Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

    Directory of Open Access Journals (Sweden)

    D.C.A. Leite

    2014-01-01

    Full Text Available Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit, the PowerSoil® DNA Isolation Kit (PS kit and the ZR Soil Microbe DNA Kit MiniprepTM (ZR kit, for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples.

  7. Comparison of DNA extraction protocols for microbial communities from soil treated with biochar

    Science.gov (United States)

    Leite, D.C.A.; Balieiro, F.C.; Pires, C.A.; Madari, B.E.; Rosado, A.S.; Coutinho, H.L.C.; Peixoto, R.S.

    2014-01-01

    Many studies have evaluated the effects of biochar application on soil structure and plant growth. However, there are very few studies describing the effect of biochar on native soil microbial communities. Microbial analysis of environmental samples requires accurate and reproducible methods for the extraction of DNA from samples. Because of the variety among microbial species and the strong adsorption of the phosphate backbone of the DNA molecule to biochar, extracting and purifying high quality microbial DNA from biochar-amended soil is not a trivial process and can be considerably more difficult than the extraction of DNA from other environmental samples. The aim of this study was to compare the relative efficacies of three commercial DNA extraction kits, the FastDNA® SPIN Kit for Soil (FD kit), the PowerSoil® DNA Isolation Kit (PS kit) and the ZR Soil Microbe DNA Kit Miniprep™ (ZR kit), for extracting microbial genomic DNA from sand treated with different types of biochar. The methods were evaluated by comparing the DNA yields and purity and by analysing the bacterial and fungal community profiles generated by PCR-DGGE. Our results showed that the PCR-DGGE profiles for bacterial and fungal communities were highly affected by the purity and yield of the different DNA extracts. Among the tested kits, the PS kit was the most efficient with respect to the amount and purity of recovered DNA and considering the complexity of the generated DGGE microbial fingerprint from the sand-biochar samples. PMID:24948928

  8. High Efficiency DNA Extraction by Graphite Oxide/Cellulose/Magnetite Composites Under Na+ Free System

    Science.gov (United States)

    Akceoglu, Garbis Atam; Li, Oi Lun; Saito, Nagahiro

    2016-04-01

    DNA extraction is the key step at various research areas like biotechnology, diagnostic development, paternity determination, and forensic science . Solid support extraction is the most common method for DNA purification. In this method, Na+ ions have often been applied as binding buffers in order to obtain high extraction efficiency and high quality of DNA; however, the presence of Na+ ions might be interfering with the downstream DNA applications. In this study, we proposed graphite oxide (GO)/magnetite composite/cellulose as an innovative material for Na+-free DNA extraction. The total wt.% of GO was fixed at 4.15% in the GO/cellulose/magnetite composite . The concentration of magnetite within the composites were controlled at 0-3.98 wt.%. The extraction yield of DNA increased with increasing weight percentage of magnetite. The highest yield was achieved at 3.98 wt.% magnetite, where the extraction efficiency was reported to be 338.5 ng/µl. The absorbance ratios between 260 nm and 280 nm (A260/A280) of the DNA elution volume was demonstrated as 1.81, indicating the extracted DNA consisted of high purity. The mechanism of adsorption of DNA was provided by (1) π-π interaction between the aromatic ring in GO and nucleobases of DNA molecule, and (2) surface charge interaction between the positive charge magnetite and anions such as phosphates within the DNA molecules. The results proved that the GO/cellulose/magnetite composite provides a Na+-free method for selective DNA extraction with high extraction efficiency of pure DNA.

  9. Automated extraction of DNA and PCR setup using a Tecan Freedom EVO® liquid handler

    DEFF Research Database (Denmark)

    Frøslev, Tobias Guldberg; Hansen, Anders Johannes; Stangegaard, Michael

    . With the Identifiler kit, the number of full DNA profiles was approximately 20% higher with DNA prepared with the robot compared to that obtained with DNA prepared manually with the Chelex method. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic...... genetic DNA typing can be implemented on a simple robot leading to the reduction of manual work as well as increased quality and throughput....

  10. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer.

    Science.gov (United States)

    Beránek, Martin; Sirák, Igor; Vošmik, Milan; Petera, Jiří; Drastíková, Monika; Palička, Vladimír

    The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

  11. The critical role of DNA extraction for detection of mycobacteria in tissues.

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    Nicolas Radomski

    Full Text Available BACKGROUND: Nucleic acid-based methods offer promise for both targeted and exploratory investigations of microbes in tissue samples. As the starting material for such studies is a mixture of host and microbial DNA, we have critically evaluated the DNA extraction step to determine the quantitative and qualitative parameters that permit faithful molecular detection of mycobacteria in infected tissue. Specifically, we assessed: 1 tissue disruption procedures; 2 DNA extraction protocols; and 3 inhibition of bacterial PCR by host DNA. PRINCIPAL FINDINGS: Regarding DNA extraction, we found that 1 grinding was not necessary if bead-beating is done, 2 the reference mycobacterial DNA extraction method recovered more pure DNA than commercial spin column kits, 3 lysozyme digestion of 1 hour was sufficient, and 4 repeated steps of phenol:chloroform:isoamyl alcohol offered minimal gain in DNA quality. By artificially mixing mycobacterial DNA with DNA extracted from uninfected mice, we found that bacterial real-time quantitative PCR was only reliable when the quantity of host DNA was < 3 µg in a final volume of 25 µl and the quality was high (260/280 nm ratio = 1.89 ± 0.08. Findings from spiked DNA studies were confirmed using DNA extracted from mice infected with different intracellular pathogens (M. tuberculosis, M. avium subsp. paratuberculosis. CONCLUSIONS: Our findings point to the most appropriate methods for extracting DNA from tissue samples for the purpose of detecting and quantifying mycobacteria. These data also inform on the limits of detection for two mycobacterial species and indicate that increasing the sample mass to improve analytic sensitivity comes at the cost of inhibition of PCR by host DNA.

  12. PCR-荧光法与 PCR-膜杂交法在检测 HPV-DNA 中的比较分析%The comparative analysis on the detection of HPV-DNA by PCR-fluorescence and PCR-membrane hybridization

    Institute of Scientific and Technical Information of China (English)

    赵一琳; 崔映红; 黄少芝

    2016-01-01

    Objective To explore different application of PCR‐fluorescence method and PCR‐hybridization method in the diag‐nosis of human papilloma virus (HPV ) infection .Methods Clinical samples were collected and detected by PCR‐ fluorescence method and PCR‐hybridization method respectively .The cell histological diagnosis was used to compare the positive rate of HPV‐DNA detection between the two methods ,and the difference was analyzed by statistical method .The specimens were divided into no intraepithelial lesion or malignancy(NILM) group(152 cases) ,atypical squamous cell hyperplasia of unknown significance(ASC‐H) group (27 cases) ,low -grade squamous intraepithelial lesion(LSIL) group(21 cases) ,high-grade squamous intraepithelial lesion (HSIL) group (2 cases) and squamous cell cervical carcinoma(SCC) group (2 cases) .Results Among 204 cases of specimens ,the results of PCR‐fluorescence showed 67 cases were positive ,137 cases were negative ,the positive rate was 32 .8% .The detection re‐sults of PCR‐membrane hybridization showed 94 cases were positive ,110 cases were negative ,the positive rate was 46 .1% .52 ca‐ses with abnormal cells were found by using cytological examination ,the proportion is 25 .5% .Within NILM group PCR‐fluores‐cence method and PCR‐membrane hybridization HPV positive rates were 12 .5% and 28 .3% respectively ,the difference was statis‐tically significant(P0 .05) .At the same time ,the difference was statistically significant between the positive rate of ab‐normal HPV cell group and the NILM group(P<0 .05) .Conclusion HPV‐DNA detected by PCR are specific and sensitive .During clinical diagnosis and treatment ,PCR‐fluorescence method has higher application value ,while in the detection and research of low -risk HPV ,PCR membrane hybridization has more advantages .The detection of HPV‐DNA combined with cell histological diagnosis can improve the diagnostic accuracy of HPV viral infection .%目的:比较并

  13. Immortalization capacity of HPV types is inversely related to chromosomal instability

    DEFF Research Database (Denmark)

    Schütze, Denise M; Krijgsman, Oscar; Snijders, Peter J F

    2016-01-01

    High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality....... This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs.Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied.......All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed...

  14. Comparison of commercial DNA extraction kits for isolation and purification of bacterial and eukaryotic DNA from PAH-contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Mahmoudi, Nagissa; Slater, Greg F.; Fulthorpe, Roberta R.

    2011-07-01

    Polycyclic aromatic hydrocarbons (PAHs) are in the environment and are carcinogenic, teratogenic and mutagenic. Their hydrophobic structure gives them low water solubility and makes them readily absorbed onto soils and sediments, where they persists until they are degraded. Microbial degradation of PAHs has been well documented and is thought to be an important process in remediating contaminated sediments and soils. Obtaining high quality purified DNA is an essential requirement for the successful DNA amplifications that underlie all subsequent procedures. Several commercial DNA extraction kits exist that provide consistent solutions for the central problems - cell lysis and humic acid removal. This study compared four commercial DNA extraction kits to extract pure, high quality bacterial and eukaryotic DNA from PAH contaminated soils and concluded that they can be used on a wide variety of soils, including heavily contaminated soils. The PowerSoil kit was the most effective and reliable.

  15. Infection with high-risk HPV types among female sex workers in northern Vietnam.

    Science.gov (United States)

    Hoang, Huyen Thi Thanh; Ishizaki, Azumi; Nguyen, Cuong Hung; Tran, Vuong Thi; Matsushita, Kaori; Saikawa, Kunikazu; Hosaka, Norimitsu; Pham, Hung Viet; Bi, Xiuqiong; Ta, Van Thanh; Van Pham, Thuc; Ichimura, Hiroshi

    2013-02-01

    Vaccines against two high-risk human papillomavirus (HPV) types, HPV-16, and HPV-18, are in use currently, with high efficacy for preventing infections with these HPV types and consequent cervical cancers. However, circulating HPV types can vary with geography and ethnicity. The aim of this study was to investigate the prevalence of HPV types and the association between HPV types and abnormal cervical cytology among female sex workers in Northern Vietnam. Cervical swabs and plasma samples were collected from 281 female sex workers at two health centers in Hanoi and Hai Phong in 2009. The HPV L1 gene was amplified by PCR using original and modified GP5(+)/6(+) primers. Amplified PCR products were genotyped by the microarray system GeneSquare (KURABO) and/or clonal sequencing. Of the 281 women, 139 (49.5%) were positive for HPV DNA. Among the HPV-positive samples, 339 strains and 29 different types were identified. Multiple-type and high risk-type HPV infections were found in 85 (61.2%) and 124 (89.2%) women, respectively. The most common genotype was HPV-52, followed by HPV-16, HPV-18, and HPV-58. Abnormal cervical cytology was detected in 3.2% (9/281) of the women, and all of these samples were positive for HPV-DNA. Age ≤25 years and infection with human immunodeficiency virus were associated positively with HPV infection among the women while ever smoking was associated negatively. These results show that HPV-52 is most prevalent among female sex workers in Northern Vietnam, most of whom had normal cervical cytology. This information may be important for designing vaccination strategies in Vietnam.

  16. Prevalence of type-specific HPV infection in Uruguay.

    Science.gov (United States)

    Berois, Nora; Heard, Isabelle; Fort, Zoraida; Alonso, Rafael; Sica, Adela; Moerzinger, Patricia; Rodriguez, Guillermo; Sancho-Garnier, Hélène; Osinaga, Eduardo; Favre, Michel

    2014-04-01

    The aim of this work was to describe the prevalence of type-specific Human papillomavirus (HPV) infection in women attending organized cervical cancer screening program in Uruguay. Nine hundred sixty-five liquid cervical cell samples obtained after collection of cervical smears for cytology were assessed for HPV DNA using the Papillocheck system (Greiner BioOne). The overall prevalence of High-Risk (HR) HPV infections was 20.8% and increased from 16.5% in women with normal cytology to 93.3% in HSIL. Prevalence of HPV 16 and/or 18 was 6.3% and HPV 16 was the most prevalent genotype in normal cytology (3.6%). The five most prevalent genotypes were HPV 16, 31, 51, 56, and 39. The overall prevalence peaked below age 30. This study provides essential baseline information at national level on type-specific HPV prevalence in Uruguay before the introduction of HPV vaccination. It documents the current prevalence of each of the oncogenic genotypes in a population attending cervical cancer screening program, suggesting that at least 64.7% of high risk lesions are potentially preventable by available HPV vaccines, and possibly augmentable if cross-protection against non-vaccine HPV types 31, 33, and 45 is confirmed. © 2013 Wiley Periodicals, Inc.

  17. A high-throughput, high-quality plant genomic DNA extraction protocol.

    Science.gov (United States)

    Li, H; Li, J; Cong, X H; Duan, Y B; Li, L; Wei, P C; Lu, X Z; Yang, J B

    2013-10-15

    The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 µg high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/µL). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R(2) = 0.9967) than the phenol-chloroform protocol (R(2) = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications.

  18. Evaluation of Five Methods for Total DNA Extraction from Western Corn Rootworm Beetles

    Science.gov (United States)

    Chen, Hong; Rangasamy, Murugesan; Tan, Sek Yee; Wang, Haichuan; Siegfried, Blair D.

    2010-01-01

    Background DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction. Methodology/Principal Findings From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol® reagent, Puregene® solutions and DNeasy® column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue) at much lower cost and less degradation as revealed on agarose gels. The DNeasy® kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle's genome, and all samples showed successful amplifications. Conclusion/Significance These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis. PMID:20730102

  19. Evaluation of five methods for total DNA extraction from western corn rootworm beetles.

    Directory of Open Access Journals (Sweden)

    Hong Chen

    Full Text Available BACKGROUND: DNA extraction is a routine step in many insect molecular studies. A variety of methods have been used to isolate DNA molecules from insects, and many commercial kits are available. Extraction methods need to be evaluated for their efficiency, cost, and side effects such as DNA degradation during extraction. METHODOLOGY/PRINCIPAL FINDINGS: From individual western corn rootworm beetles, Diabrotica virgifera virgifera, DNA extractions by the SDS method, CTAB method, DNAzol reagent, Puregene solutions and DNeasy column were compared in terms of DNA quantity and quality, cost of materials, and time consumed. Although all five methods resulted in acceptable DNA concentrations and absorbance ratios, the SDS and CTAB methods resulted in higher DNA yield (ng DNA vs. mg tissue at much lower cost and less degradation as revealed on agarose gels. The DNeasy kit was most time-efficient but was the costliest among the methods tested. The effects of ethanol volume, temperature and incubation time on precipitation of DNA were also investigated. The DNA samples obtained by the five methods were tested in PCR for six microsatellites located in various positions of the beetle's genome, and all samples showed successful amplifications. CONCLUSION/SIGNIFICANCE: These evaluations provide a guide for choosing methods of DNA extraction from western corn rootworm beetles based on expected DNA yield and quality, extraction time, cost, and waste control. The extraction conditions for this mid-size insect were optimized. The DNA extracted by the five methods was suitable for further molecular applications such as PCR and sequencing by synthesis.

  20. DNA extraction protocols from dormant buds of twelve woody plant genera

    Science.gov (United States)

    Standard plant DNA extraction protocols call for samples of newly expanding leaves and shoots yet analysis is sometimes needed when plants are dormant. We evaluated three DNA extraction protocols using dormant buds from 40 species and four hybrids of 12 genera. Two protocols were from ready-to-use ...

  1. A RAPID DNA EXTRACTION METHOD FOR PCR IDENTIFICATION OF FUNGAL INDOOR AIR CONTAMINANTS

    Science.gov (United States)

    Following air sampling, fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and polymerase chain reaction (PCR) appli...

  2. A RAPID DNA EXTRACTION METHOD FOR PCR IDENTIFICATION OF FUNGAL INDOOR AIR CONTAMINANTS

    Science.gov (United States)

    Following air sampling, fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and polymerase chain reaction (PCR) appli...

  3. Suitability of the boiling method of DNA extraction in mosquitoes for routine molecular analyses

    Directory of Open Access Journals (Sweden)

    D. K. Sarma

    2014-09-01

    Full Text Available This communication deals with the experience on suitability of the boiling method of DNA extraction from mosquito tissues. The DNA extracted by this method was found, by and large, stable after 30 months of storage. The method is useful for routine molecular entomological applications.

  4. Evaluation of primary HPV-DNA testing in relation to visual inspection methods for cervical cancer screening in rural China: an epidemiologic and cost-effectiveness modelling study

    Directory of Open Access Journals (Sweden)

    Kang Yoon-Jung

    2011-06-01

    Full Text Available Abstract Background A new lower-cost rapid-throughput human papillomavirus (HPV test (careHPV, Qiagen, Gaithersburg, USA has been shown to have high sensitivity for the detection of high grade cervical intraepithelial neoplasia. Methods We assessed the outcomes and cost-effectiveness of careHPV screening in rural China, compared to visual inspection with acetic acid, when used alone (VIA or in combination with Lugol's iodine (VIA/VILI. Using data on sexual behaviour, test accuracy, diagnostic practices and costs from studies performed in rural China, we estimated the cost-effectiveness ratio (CER and associated lifetime outcomes for once-lifetime and twice-lifetime screening strategies, and for routine screening at 5-yearly, 10-yearly and IARC-recommended intervals. The optimal age range for once-lifetime screening was also assessed. Results For all strategies, the relative ordering of test technologies in reducing cervical cancer incidence and mortality was VIA (least effective; VIA/VILI; careHPV@1.0 pg/ml and careHPV@0.5 pg/ml (most effective. For once-lifetime strategies, maximum effectiveness was achieved if screening occurred between 35-50 years. Assuming a participation rate of ~70%, once-lifetime screening at age 35 years would reduce cancer mortality by 8% (for VIA to 12% (for careHPV@0.5 over the long term, with a CER of US$557 (for VIA to $959 (for careHPV@1.0 per life year saved (LYS compared to no intervention; referenced to a 2008 GDP per capita in Shanxi Province of $2,975. Correspondingly, regular screening with an age-standardised participation rate of 62% (which has been shown to be achievable in this setting would reduce cervical cancer mortality by 19-28% (for 10-yearly screening to 43-54% (using IARC-recommended intervals, with corresponding CERs ranging from $665 (for 10-yearly VIA to $2,269 (for IARC-recommended intervals using careHPV@1.0 per LYS. Conclusions This modelled analysis suggests that primary careHPV screening

  5. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

    Science.gov (United States)

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  6. Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hasseldam; Andersen, Sandra Christine; Christensen, Julia

    2015-01-01

    The efficiency of ten widely applied DNA extraction protocols was evaluated for suitability for diagnostic metagenomics. The protocols were selected based on a thorough literature study. Chicken fecal samples inoculated with about 1×103 and 1×106CFU/g Campylobacter jejuni were used as a model...... of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA........ The evaluation was performed based on total DNA yield measured by fluorometry, and quality and quantity of C. jejuni DNA measured by real-time PCR. There was up to a 25-fold variance between the lowest (NucliSens miniMAG, BIOMÉRIEUX) and highest (PowerLyzer PowerSoil DNA Isolation Kit, MO BIO Laboratories...

  7. COMPARISON OF COMMERCIAL DNA KITS AND TRADITIONAL DNA EXTRACTION PROCEDURE IN PCR DETECTION OF PORK IN DRY/FERMENTED SAUSAGES

    Directory of Open Access Journals (Sweden)

    Ivona Djurkin Kušec

    2015-09-01

    Full Text Available In the present study four commercially available DNA extraction kits (Wizard® Genomic DNA Purification Kit, High Pure PCR Template Kit, DNeasy mericon Food and GeneJET PCR Purification Kit, as well as standard phenol/chloroform isolation technique have been evaluated regarding their concentration, purity and suitability for amplification of porcine DNA in dry/fermented sausages. The isolates were assessed for quantity and quality using spectrophotometer (IMPLEN GmbH, Germany. To verify template usability and quality of isolated DNA, the polymerase chain reaction (PCR targeting at porcine cytochrome b by species specific primers was used. The comparison of extraction methods revealed satisfactory efficiency and purity of all extraction kits, while with standard phenol/chloroform isolation method high concentrations of DNA with low A260/280 were obtained. However, all the investigated techniques proved to be suitable for identification of porcine DNA in dry/fermented sausage. Thus, the standard phenol/chloroform DNA extraction method, as the cost-effective one, can be recommended as a good alternative to more expensive isolation kits when investigating the presence of pork DNA in dry/ fermented meat products.

  8. La baja utilidad de la determinación del ADN del VPH en la región distal de la uretra masculina Low usefulness of HPV DNA determination in the distal region of the male urethra

    Directory of Open Access Journals (Sweden)

    Ahideé G Leyva-López

    2003-01-01

    Full Text Available OBJETIVO: Determinar la prevalencia uretral del ácido desoxirribonucleico del virus de papiloma humano, condilomatosis clínica y subclínica, en hombres cuyas parejas sexuales tuvieron el antecedente de neoplasia intraepitelial cervical. MATERIAL Y MÉTODOS: De octubre de 1997 a agosto de 1998 se hizo un estudio transversal; se incluyeron 200 hombres de entre 17 a 64 años de edad, referidos a la Coordinación de Oncología del Instituto Nacional de Perinatología, de la Ciudad de México, porque sus parejas regulares sexuales tuvieron el antecedente de neoplasia intraepitelial cervical. Se llevó a cabo un examen físico del pene (penoscopía con la aplicación de ácido acético a 3-5%, y con el uso de un colposcopio se localizaron y evaluaron zonas acetoblancas y cambios vasculares, interpretados como anormales, asociados con la infección por el virus del papiloma humano. La determinación del ADN de VPH se verificó por PCR e hibridación en línea reversa. El análisis estadístico exploratorio y univariante se realizó con el paquete Stata V6.0. RESULTADOS: En las 200 muestras recolectadas de células exfoliadas de la uretra el gen de beta-globina estuvo presente en 93.5% (187/200, y el ácido desoxirribonucleico del virus del papiloma humano fue detectable solamente en 2% (4/187 de los sujetos. Por medio de la penoscopía se observó la presencia de zonas acetoblancas en 43% (81/187 de los sujetos. CONCLUSIONES: En este estudio se observa que la presencia del ácido desoxirribonucleico del virus del papiloma humano en la uretra masculina es poco común, como lo reportan estudios internacionales. Es necesario realizar investigaciones que evalúen esta presencia en glande y surco balano prepucial, en comparación con la región distal de la uretra.OBJECTIVE: To assess the prevalence of Human Papillomavirus (HPV Deoxyribonucleic acid (DNA, and of clinical and subclinical condilomatosis in men whose sex partners had been diagnosed with

  9. A comparative study of two methods for the isolation of human leucocytes for DNA extraction.

    Science.gov (United States)

    Lim, L H; Ton, S H; Cheong, S K

    1990-06-01

    The 'Dextran' and the 'Buffy-coat' methods for isolation of human leucocytes for DNA extraction were compared on the basis of DNA yield from the same amounts (10 ml) of blood. Human leucocytes from a total of 11 samples were isolated using both methods for each sample after which DNA was extracted. Extracted DNA samples were treated with ribonucleases and proteinase K after which the yields were quantitated by measuring absorbance at 260 nm. The 'Buffy-coat' method yielded a mean concentration of DNA of 476.7 micrograms/ml (range: 212 to 700 micrograms/ml) while the 'Dextran' method yielded 188.4 micrograms/ml (range: 64 to 340 micrograms/ml). The difference was confirmed by subjecting the extracted DNA samples to agarose gel electrophoresis.

  10. An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

    Science.gov (United States)

    Verma, Digvijay; Satyanarayana, T

    2011-09-01

    An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.

  11. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction

    Science.gov (United States)

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol–chloroform–isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction. PMID:24834966

  12. The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol-chloroform-isoamyl alcohol DNA extraction.

    Science.gov (United States)

    Renshaw, Mark A; Olds, Brett P; Jerde, Christopher L; McVeigh, Margaret M; Lodge, David M

    2015-01-01

    Current research targeting filtered macrobial environmental DNA (eDNA) often relies upon cold ambient temperatures at various stages, including the transport of water samples from the field to the laboratory and the storage of water and/or filtered samples in the laboratory. This poses practical limitations for field collections in locations where refrigeration and frozen storage is difficult or where samples must be transported long distances for further processing and screening. This study demonstrates the successful preservation of eDNA at room temperature (20 °C) in two lysis buffers, CTAB and Longmire's, over a 2-week period of time. Moreover, the preserved eDNA samples were seamlessly integrated into a phenol-chloroform-isoamyl alcohol (PCI) DNA extraction protocol. The successful application of the eDNA extraction to multiple filter membrane types suggests the methods evaluated here may be broadly applied in future eDNA research. Our results also suggest that for many kinds of studies recently reported on macrobial eDNA, detection probabilities could have been increased, and at a lower cost, by utilizing the Longmire's preservation buffer with a PCI DNA extraction. © 2014 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

  13. Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

    Science.gov (United States)

    Kathiravan, Mathur Nadarajan; Gim, Geun Ho; Ryu, Jaewon; Kim, Pyung Il; Lee, Chul Won; Kim, Si Wouk

    2015-11-01

    In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples.

  14. How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

    NARCIS (Netherlands)

    Särkinen, T.; Staats, M.; Richardson, J.E.; Cowan, R.S.; Bakker, F.T.

    2012-01-01

    Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance

  15. HPV Infection in Men

    Directory of Open Access Journals (Sweden)

    Joel M. Palefsky

    2007-01-01

    Full Text Available While much is known about the natural history of cervical human papillomavirus (HPV infection and its consequences, including cervical intraepithelial neoplasia and cervical cancer, relatively little is known about the natural history of anogenital HPV infection and diseases in men. In part this reflects difficulties in penile sampling and visual assessment of penile lesions. Anal HPV infection and disease also remain poorly understood. Although HPV is transmitted sexually and infects the genitals of both sexes, the cervix remains biologically more vulnerable to malignant transformation than does the penis or anus in men. An understanding of male HPV infection is therefore important in terms of reducing transmission of HPV to women and improving women's health. However, it is also important due to the burden of disease in men, who may develop both penile and anal cancer, particularly among HIV-positive men who have sex with men. Improved sampling techniques of the male genitalia and cohort studies in progress should provide important information on the natural history of anogenital HPV infection and disease in men, including risk factors for HPV acquisition and transmission. The impact of HPV vaccination in women on male anogenital HPV infection will also need to be assessed.

  16. Soil DNA Extraction Procedure Influences Protist 18S rRNA Gene Community Profiling Outcome

    DEFF Research Database (Denmark)

    Santos, Susana S; Nunes, Inês; Nielsen, Tue Kjærgaard

    2017-01-01

    Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two ma...... high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays....

  17. Efficiency comparison of three methods for extracting genomic DNA of the pathogenic oomycete Pythium insidiosum.

    Science.gov (United States)

    Lohnoo, Tassanee; Jongruja, Nujarin; Rujirawat, Thidarat; Yingyon, Wanta; Lerksuthirat, Tassanee; Nampoon, Umporn; Kumsang, Yothin; Onpaew, Pornpit; Chongtrakool, Piriyaporn; Keeratijarut, Angsana; Brandhorst, Tristan T; Krajaejun, Theerapong

    2014-03-01

    The fungus-like organism Pythium insidiosum is the causative agent of a life-threatening tropical infectious disease, pythiosis, which has high rates of morbidity and mortality. A lack of reliable diagnostic tools and effective treatments for pythiosis presents a major challenge to healthcare professionals. Unfortunately, surgical removal of infected organs remains the default treatment for pythiosis. P. insidiosum is an understudied organism. In-depth study of the pathogen at the molecular level could lead to better means of infection control High quality genomic DNA (gDNA) is needed for molecular biology-based research and application development, such as: PCR-assisted diagnosis, population studies, phylogenetic analysis, and molecular genetics assays. To evaluate quality and quantity of the P. insidiosum gDNA extracted by three separate protocols intended for fungal gDNA preparation. Seven P. insidiosum isolates were subjected to gDNA extraction by using conventional-extraction, rapid-extraction, and salt-extraction protocols. The conventional protocol offered the best gDNA in terms of quality and quantity, and could be scaled up. The rapid-extraction protocol had a short turnaround time, but the quality and quantity of the gDNA obtained were limited. The salt-extraction protocol was simple, rapid, and efficient, making it appealing for high throughput preparation of small-scale gDNA samples. Compared to rapid-extraction protocol, both conventional-extraction and salt-extraction protocols provided a better quality and quantity of gDNA, suitable for molecular studies of P. insidiosum. In contrast to the other two methods, the salt-extraction protocol does not require the use of hazardous and expensive materials such as phenol, chloroform, or liquid nitrogen.

  18. Clinical value of TCT combined with HPV-DNA detection in cervical cancer screening%液基薄层细胞学联合 HPV-DNA检测对宫颈癌患者筛查的临床价值

    Institute of Scientific and Technical Information of China (English)

    李媛媛

    2016-01-01

    Objective:To explore clinical value ThinPrep cytology test ( TCT) and HPV detection in cervical cancer screen-ing. Methods:The clinical data of the patients in the screening of cervical precancerous lesions were collected. Each patient was given TCT combined with HPV detection. Finally, after colposcopy cervical biopsy, the sensitivity, accuracy and false positive rate of single TCT, single HPV and TCT combined with HPV in the diagnosis of chronic cervical inflammation, cervical precancerous lesions and cervical cancer were compared. Results:The cervical cancer and precancerous lesions screening under colposcopy biopsy was done in 360 patients. Among the 3 screening methods, the combined application of TCT and HPV had the highest sensitivity and specificity, and could be used for diagnosing severe atypical hyperplasia ( CIN-III) and cervical carcinoma without missed diagnosis;however, it might miss some moderate dysplasia ( CIN-II) and mild atypical hyperplasia ( CIN-I) patients. Conclusions:The TCT combined with HPV-DNA detection can be used actively in the cervical precancerous lesions screening, and improve the accuracy of clinical diagno-sis. Moreover, combined with colposcopy biopsy of cervical cancer and precancerous lesions, it can provide a diagnostic basis for the clinical treatment, and is worthy of clinical application.%目的::探讨液基薄层细胞学与HPV检测联合应用后在宫颈癌患者筛查中的临床应用价值。方法:分析行宫颈癌前病变筛查的患者共360例临床资料,每位患者给予薄层液基细胞学联合HPV检测,最后经阴道镜下宫颈活检,比较单独TCT检查、单独HPV检查及两种检查联合检测在诊断患者宫颈慢性炎症、宫颈癌前病变及宫颈癌的敏感程度、准确性和假阳性率。结果:宫颈癌及癌前病变筛查患者,行阴道镜下活检的患者360例。3种筛选方法中,联合应用TCT及HPV的敏感性、特异性最

  19. PCR-fingerprint profiles of mitochondrial and genomic DNA extracted from Fetus cervi using different extraction methods.

    Science.gov (United States)

    Ai, Jinxia; Wang, Xuesong; Gao, Lijun; Xia, Wei; Li, Mingcheng; Yuan, Guangxin; Niu, Jiamu; Zhang, Lihua

    2016-06-01

    The use of Fetus cervi, which is derived from the embryo and placenta of Cervus Nippon Temminck or Cervs elaphus Linnaeus, has been documented for a long time in China. There are abundant species of deer worldwide. Those recorded by China Pharmacopeia (2010 edition) from all the species were either authentic or adulterants/counterfeits. Identification of their origins or authenticity became a key in the preparation of the authentic products. The traditional SDS alkaline lysis and salt-outing methods were modified to extract mt DNA and genomic DNA from fresh and dry Fetus cervi in addition to Fetus from false animals, respectively. A set of primers were designed by bioinformatics to target the intra-and inter-variation. The mt DNA and genomic DNA extracted from Fetus cervi using the two methods meet the requirement for authenticity. Extraction of mt DNA by SDS alkaline lysis is more practical and accurate than extraction of genomic DNA by salt-outing method. There were differences in length and number of segments amplified by PCR between mt DNA from authentic Fetus cervi and false animals Fetus. The distinctive PCR-fingerprint patterns can distinguish the Fetus cervi from adulterants and counterfeit animal Fetus.

  20. Comparison of Three Different DNA Extraction Methods for Linguatula serrata as a Food Born Pathogen.

    Science.gov (United States)

    Eslami, Gilda; Khalatbari-Limaki, Sepideh; Ehrampoush, Mohammad Hasan; Gholamrezaei, Mostafa; Hajimohammadi, Bahador; Oryan, Ahmad

    2017-01-01

    One of the most important items in molecular characterization of food-borne pathogens is high quality genomic DNA. In this study, we investigated three protocols and compared their simplicity, duration and costs for extracting genomic DNA from Linguatula serrata. The larvae were collected from the sheep's visceral organs from the Yazd Slaughterhouse during May 2013. DNA extraction was done in three different methods, including commercial DNA extraction kit, Phenol Chloroform Isoamylalcohol (PCI), and salting out. Extracted DNA in each method was assessed for quantity and quality using spectrophotometery and agarose gel electrophoresis, respectively. The less duration was regarding to commercial DNA extraction kit and then salting out protocol. The cost benefit one was salting out and then PCI method. The best quantity was regarding to PCI with 72.20±29.20 ng/μl, and purity of OD260/OD280 in 1.76±0.947. Agarose gel electrophoresis for assessing the quality found all the same. Salting out is introduced as the best method for DNA extraction from L. seratta as a food-borne pathogen with the least costand appropriate purity. Although, the best purity was regarding to PCI but PCI is not safe as salting out. In addition, the duration of salting out was less than PCI. The least duration was seen in commercial DNA extraction kit, but it is expensive and therefore is not recommended for developing countries where consumption of offal is common.

  1. Evaluation of methods for the extraction of DNA from drinking water distribution system biofilms.

    Science.gov (United States)

    Hwang, Chiachi; Ling, Fangqiong; Andersen, Gary L; LeChevallier, Mark W; Liu, Wen-Tso

    2012-01-01

    While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories.

  2. Extraction of human genomic DNA from whole blood using a magnetic microsphere method.

    Science.gov (United States)

    Gong, Rui; Li, Shengying

    2014-01-01

    With the rapid development of molecular biology and the life sciences, magnetic extraction is a simple, automatic, and highly efficient method for separating biological molecules, performing immunoassays, and other applications. Human blood is an ideal source of human genomic DNA. Extracting genomic DNA by traditional methods is time-consuming, and phenol and chloroform are toxic reagents that endanger health. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA. In this study, we developed urea-formaldehyde resin magnetic microspheres and magnetic silica microspheres for extraction of human genomic DNA. First, a magnetic microsphere suspension was prepared and used to extract genomic DNA from fresh whole blood, frozen blood, dried blood, and trace blood. Second, DNA content and purity were measured by agarose electrophoresis and ultraviolet spectrophotometry. The human genomic DNA extracted from whole blood was then subjected to polymerase chain reaction analysis to further confirm its quality. The results of this study lay a good foundation for future research and development of a high-throughput and rapid extraction method for extracting genomic DNA from various types of blood samples.

  3. Extraction of DNA from honey and its amplification by PCR for botanical identification

    Directory of Open Access Journals (Sweden)

    Sona Arun Jain

    2013-12-01

    Full Text Available The physiochemical and biological properties of honey are directly associated to its floral origin. Some current commonly used methods for identification of botanical origin of honey involve palynological analysis, chromatographic methods, or direct observation of the bee behavior. However, these methods can be less sensitive and time consuming. DNA-based methods have become popular due to their simplicity, quickness, and reliability. The main objective of this research is to introduce a protocol for the extraction of DNA from honey and demonstrate that the molecular analysis of the extracted DNA can be used for its botanical identification. The original CTAB-based protocol for the extraction of DNA from plants was modified and used in the DNA extraction from honey. DNA extraction was carried out from different honey samples with similar results in each replication. The extracted DNA was amplified by PCR using plant specific primers, confirming that the DNA extracted using the modified protocol is of plant origin and has good quality for analysis of PCR products and that it can be used for botanical identification of honey.

  4. Apoptotic HPV positive cancer cells exhibit transforming properties.

    Directory of Open Access Journals (Sweden)

    Emilie Gaiffe

    Full Text Available Previous studies have shown that DNA can be transferred from dying engineered cells to neighboring cells through the phagocytosis of apoptotic bodies, which leads to cellular transformation. Here, we provide evidence of an uptake of apoptotic-derived cervical cancer cells by human mesenchymal cells. Interestingly, HeLa (HPV 18+ or Ca Ski (HPV16+ cells, harboring integrated high-risk HPV DNA but not C-33 A cells (HPV-, were able to transform the recipient cells. Human primary fibroblasts engulfed the apoptotic bodies effectively within 30 minutes after co-cultivation. This mechanism is active and involves the actin cytoskeleton. In situ hybridization of transformed fibroblasts revealed the presence of HPV DNA in the nucleus of a subset of phagocytosing cells. These cells expressed the HPV16/18 E6 gene, which contributes to the disruption of the p53/p21 pathway, and the cells exhibited a tumorigenic phenotype, including an increased proliferation rate, polyploidy and anchorage independence growth. Such horizontal transfer of viral oncogenes to surrounding cells that lack receptors for HPV could facilitate the persistence of the virus, the main risk factor for cervical cancer development. This process might contribute to HPV-associated disease progression in vivo.

  5. Optimized DNA extraction from neonatal dried blood spots: application in methylome profiling.

    Science.gov (United States)

    Ghantous, Akram; Saffery, Richard; Cros, Marie-Pierre; Ponsonby, Anne-Louise; Hirschfeld, Steven; Kasten, Carol; Dwyer, Terence; Herceg, Zdenko; Hernandez-Vargas, Hector

    2014-07-01

    Neonatal dried blood spots (DBS) represent an inexpensive method for long-term biobanking worldwide and are considered gold mines for research for several human diseases, including those of metabolic, infectious, genetic and epigenetic origin. However, the utility of DBS is restricted by the limited amount and quality of extractable biomolecules (including DNA), especially for genome wide profiling. Degradation of DNA in DBS often occurs during storage and extraction. Moreover, amplifying small quantities of DNA often leads to a bias in subsequent data, particularly in methylome profiles. Thus it is important to develop methodologies that maximize both the yield and quality of DNA from DBS for downstream analyses. Using combinations of in-house-derived and modified commercial extraction kits, we developed a robust and efficient protocol, compatible with methylome studies, many of which require stringent bisulfite conversion steps. Several parameters were tested in a step-wise manner, including blood extraction, cell lysis, protein digestion, and DNA precipitation, purification and elution. DNA quality was assessed based on spectrophotometric measurements, DNA detectability by PCR, and DNA integrity by gel electrophoresis and bioanalyzer analyses. Genome scale Infinium HumanMethylation450 and locus-specific pyrosequencing data generated using the refined DBS extraction protocol were of high quality, reproducible and consistent. This study may prove useful to meet the increased demand for research on prenatal, particularly epigenetic, origins of human diseases and for newborn screening programs, all of which are often based on DNA extracted from DBS.

  6. DNA extraction technique for different rice varieties grown in Sri Lanka

    Directory of Open Access Journals (Sweden)

    Ranganathan Kapilan

    2015-09-01

    Full Text Available Extraction of DNA is very important nowadays in bio-molecular researches. Extracted DNA should be purified and the quality of DNA should also be very high. The objective of the study was to develop a simple efficient method to isolate DNA from the rice varieties in an open laboratory environment, and to eliminate the usage of expensive chemicals and tools. The DNA extraction methods developed by the DNeasy plant kit method supplied by QIAGEN, Cheng et al., Doyle et al. and Michiels et al. were applied to five different rice varieties grown in different parts of Sri Lanka. Based on the quantity and quality of the extracted DNA tested by measuring the absorbance of DNA at 260 nm using Nanodrop® ND-1000 spectrophotometer and measuring the ratio of A260 / A280 and gel running on agarose, the efficiency of the extraction method chosen varied among rice varieties. Among the methods used, the methods introduced by DNeasy plant kit method supplied by QIAGEN and Cheng et al, yielded good and amplifiable quality DNA with satisfactory concentration for all the rice varieties tested. Therefore the modified method of Cheng et al, 1987 could be used to extract DNA from rice varieties instead of the commercially available expensive and hazardous DNeasy plant kit method supplied by QIAGEN.

  7. A comparative evaluation of four DNA extraction protocols from whole blood sample.

    Science.gov (United States)

    Ghaheri, M; Kahrizi, D; Yari, K; Babaie, A; Suthar, R S; Kazemi, E

    2016-03-31

    All organisms have Deoxyribonucleic acid (DNA) within their cells. DNA is a complex molecule that contains all of the information necessary to build and maintain an organism. DNA extraction is one of the most basic and essential techniques in the study of DNA that allow huge advances in molecular biology, biotechnology and bioinformatics laboratories. Whole blood samples are one of the main sources used to obtain DNA and there are many different protocols available in this issue. In current research, compared four DNA extraction protocols from blood samples; include modified phenol-chloroform protocol, two salting-out and enzyme free method and from commercial kit. The extracted DNAs by these protocols were analyzed according to their time demands, quality and quantity, toxicity and functionality in PCR method. Also the quality and quantity of the extracted DNA were surveyed by gel electrophoresis and Nanodrop spectrophotometry methods. It was observed that there are not significantly differences between these methods about DNA Purity (A260/A280), but the DNA yield (ng DNA/μl) of phenol/chloroform method was higher than other methods. In addition, phenol/chloroform was the most toxic method and it takes more time than other methods. Roche diagnostics GmbH kit was the most expensive among the four methods but the least extraction time was required and it was the safest method.

  8. A high volume extraction and purification method for recovering DNA from human bone.

    Science.gov (United States)

    Marshall, Pamela L; Stoljarova, Monika; Schmedes, Sarah E; King, Jonathan L; Budowle, Bruce

    2014-09-01

    DNA recovery, purity and overall extraction efficiency of a protocol employing a novel silica-based column, Hi-Flow(®) (Generon Ltd., Maidenhead, UK), were compared with that of a standard organic DNA extraction methodology. The quantities of DNA recovered by each method were compared by real-time PCR and quality of DNA by STR typing using the PowerPlex(®) ESI 17 Pro System (Promega Corporation, Madison, WI) on DNA from 10 human bone samples. Overall, the Hi-Flow method recovered comparable quantities of DNA ranging from 0.8ng±1 to 900ng±159 of DNA compared with the organic method ranging from 0.5ng±0.9 to 855ng±156 of DNA. Complete profiles (17/17 loci tested) were obtained for at least one of three replicates for 3/10 samples using the Hi-Flow method and from 2/10 samples with the organic method. All remaining bone samples yielded partial profiles for all replicates with both methods. Compared with a standard organic DNA isolation method, the results indicated that the Hi-Flow method provided equal or improved recovery and quality of DNA without the harmful effects of organic extraction. Moreover, larger extraction volumes (up to 20mL) can be employed with the Hi-Flow method which enabled more bone sample to be extracted at one time. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  9. HPV detection in oral carcinomas Detecção do HPV em carcinomas orais

    Directory of Open Access Journals (Sweden)

    Aurora Karla de Lacerda Vidal

    2004-02-01

    Full Text Available The authors set out in this study to verify the presence of low- and high-risk DNA of human papillomavirus (HPV in oral cancer by means of the hybrid capture Digene® test (São Paulo-SP, Brazil in smears from exfoliative cytology and also to compare the findings with those of conventional light microscopy (hematoxylin-eosin (HE/Papanicolaou. Forty individuals gave their written informed consent to participate in the study and also had their clinical data analyzed. The 40 exfoliative cytology examinations performed to date produced the following results: 29 (72.5% negative for low- and high-risk HPV-DNA; nine (22.5% positive for low- and high-risk HPV-DNA; one (2.5% positive for low-risk HPV-DNA; and one (2.5% positive for high-risk HPV-DNA. There was agreement among the findings for the presence of DNA-HPV for both exfoliative cytology (smear to hybrid capture Digene® test and the cytological smear readings made by conventional light microscopy. It was therefore concluded that the HPV virus may be a cocarcinogen of the mouth cancer as it is in the cervix cancer.Os autores buscaram verificar, neste estudo, a presença do papilomavírus humano (HPV de baixo e de alto risco em carcinomas orais através do teste de captura híbrida Digene® (São Paulo-SP, Brasil em amostras colhidas pela citologia esfoliativa bucal e, ainda, avaliar comparativamente as referidas leituras com alterações celulares indicativas deste vírus obtidas com a interpretação citológica óptica convencional (hematoxilina-eosina (HE/Papanicolaou. Quarenta indivíduos concordaram, espontaneamente, através de assinatura do termo de consentimento livre e esclarecido, em participar da pesquisa, e seus dados clínicos foram analisados. Entre as 40 amostras provenientes da citologia esfoliativa 29 (72,5% mostraram-se negativas para presença de HPV-DNA de baixo e de alto risco; nove (22,5% foram positivas para o HPV-DNA de baixo e de alto risco; uma (2,5% foi positiva apenas

  10. Evaluation of an automated protocol for efficient and reliable DNA extraction of dietary samples.

    Science.gov (United States)

    Wallinger, Corinna; Staudacher, Karin; Sint, Daniela; Thalinger, Bettina; Oehm, Johannes; Juen, Anita; Traugott, Michael

    2017-08-01

    Molecular techniques have become an important tool to empirically assess feeding interactions. The increased usage of next-generation sequencing approaches has stressed the need of fast DNA extraction that does not compromise DNA quality. Dietary samples here pose a particular challenge, as these demand high-quality DNA extraction procedures for obtaining the minute quantities of short-fragmented food DNA. Automatic high-throughput procedures significantly decrease time and costs and allow for standardization of extracting total DNA. However, these approaches have not yet been evaluated for dietary samples. We tested the efficiency of an automatic DNA extraction platform and a traditional CTAB protocol, employing a variety of dietary samples including invertebrate whole-body extracts as well as invertebrate and vertebrate gut content samples and feces. Extraction efficacy was quantified using the proportions of successful PCR amplifications of both total and prey DNA, and cost was estimated in terms of time and material expense. For extraction of total DNA, the automated platform performed better for both invertebrate and vertebrate samples. This was also true for prey detection in vertebrate samples. For the dietary analysis in invertebrates, there is still room for improvement when using the high-throughput system for optimal DNA yields. Overall, the automated DNA extraction system turned out as a promising alternative to labor-intensive, low-throughput manual extraction methods such as CTAB. It is opening up the opportunity for an extensive use of this cost-efficient and innovative methodology at low contamination risk also in trophic ecology.

  11. Using a commercial DNA extraction kit to obtain RNA from mature rice kernels

    Science.gov (United States)

    Few RNA extraction protocols or commercial kits work well with the starchy endosperm of cereal grains. Standard RNA extraction protocols are time consuming, use large amounts of expensive chemicals, and leave behind hazardous wastes. However, there are numerous commercial DNA extraction kits that ...

  12. Bioaerosol DNA Extraction Technique from Air Filters Collected from Marine and Freshwater Locations

    Science.gov (United States)

    Beckwith, M.; Crandall, S. G.; Barnes, A.; Paytan, A.

    2015-12-01

    Bioaerosols are composed of microorganisms suspended in air. Among these organisms include bacteria, fungi, virus, and protists. Microbes introduced into the atmosphere can drift, primarily by wind, into natural environments different from their point of origin. Although bioaerosols can impact atmospheric dynamics as well as the ecology and biogeochemistry of terrestrial systems, very little is known about the composition of bioaerosols collected from marine and freshwater environments. The first step to determine composition of airborne microbes is to successfully extract environmental DNA from air filters. We asked 1) can DNA be extracted from quartz (SiO2) air filters? and 2) how can we optimize the DNA yield for downstream metagenomic sequencing? Aerosol filters were collected and archived on a weekly basis from aquatic sites (USA, Bermuda, Israel) over the course of 10 years. We successfully extracted DNA from a subsample of ~ 20 filters. We modified a DNA extraction protocol (Qiagen) by adding a beadbeating step to mechanically shear cell walls in order to optimize our DNA product. We quantified our DNA yield using a spectrophotometer (Nanodrop 1000). Results indicate that DNA can indeed be extracted from quartz filters. The additional beadbeating step helped increase our yield - up to twice as much DNA product was obtained compared to when this step was omitted. Moreover, bioaerosol DNA content does vary across time. For instance, the DNA extracted from filters from Lake Tahoe, USA collected near the end of June decreased from 9.9 ng/μL in 2007 to 3.8 ng/μL in 2008. Further next-generation sequencing analysis of our extracted DNA will be performed to determine the composition of these microbes. We will also model the meteorological and chemical factors that are good predictors for microbial composition for our samples over time and space.

  13. Standardization of DNA extraction from invasive alien weed ...

    African Journals Online (AJOL)

    DNA isolation from the weed, Parthenium hysterophorus is complicated due to the ... version of cetyltrimethylammonium bromide (CTAB) method constituting high ... The resulted genomic DNA showed fine random amplified polymorphic DNA ...

  14. Extracting DNA from 'jaws': High yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material

    DEFF Research Database (Denmark)

    Eg Nielsen, Einar; Morgan, J. A T; Maher, S. L.

    2017-01-01

    of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples...... and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield....

  15. Primary Screening for Cervical Cancer Based on High-Risk Human Papillomavirus (HPV) Detection and HPV 16 and HPV 18 Genotyping, in Comparison to Cytology

    Science.gov (United States)

    Constantinidis, Theocharis; Constantinidis, Theodoros C.

    2015-01-01

    Objectives The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening. Methods The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein. Results Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+ (sensitivity: 58.5%; specificity 97.5%) as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology). Conclusion HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18

  16. The Prevalence of Human Papilloma Virus(HPV in Malignant Cervical Lesion, Using Multiplex PCR

    Directory of Open Access Journals (Sweden)

    M. R. Keyhkhaee

    2006-07-01

    Full Text Available Background: Cervical cancer is the second leading cause of cancer death among women. In this cancer, the effects of prevention, early diagnosis and treatment more than other cancers decrease the mortality rate. In 1970 human papilloma virus (HPV was introduction as major etiologic factor of cervical cancer. Different studies throughout the world revealed strong correlation between HPV and cancerous & precancerous changes in epithelial cells. Since cell culture and serological methods can not recognize the virus and its subtypes, the importance of the molecular methods including polymerase chain reaction (PCR in early and definite diagnosis of virus is obvious. Methods: In this study, after patient selection using the related protocol and completion of the questionnaires, 100 samples from cancer lesions of cervix selected. Then DNA extraction from paraffin blocks performed using standard method. Multiplex PCR with two pairs of primer (one as internal control performed and the PCR product run on 8% polyacrylamid gel. Results: The results showed that 73% of the tissues were infected by HPV. Conclusion: This finding confirm the previous results based of correlation between HPV,and cervical cancer.

  17. Microfluidic chip for stacking, separation and extraction of multiple DNA fragments.

    Science.gov (United States)

    Wu, Ruige; Seah, Y P; Wang, Zhiping

    2016-03-11

    A disposable integrated microfluidic device was developed for rapid sample stacking, separation and extraction of multiple DNA fragments from a relatively large amount of sample. Isotachophoresis hyphenated gel electrophoresis (ITP-GE) was used to pre-concentrate and separate DNA fragments, followed by extraction of pure DNA fragments with electroelution on-chip. DNA fragments of 200bp, 500bp and 1kbp were successfully separated and collected in the extraction chamber within 25min. The extraction efficiency obtained from the chip was 49.9%, 52.1% and 53.7% for 200bp, 500bp and 1kbp DNA fragments, respectively. The extracted DNA fragments exhibited compatibility with downstream enzymatic reactions, for example PCR. The chip was also used to extract DNA fragments with specific size range from sheared genomic DNA and demonstrated similar performance to that using traditional gel cutting method. The whole assay can finish in 32min, 6 times faster than traditional method. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. DNA extraction procedure affects organic-aggregate-attached bacterial community profiles from a shallow eutrophic lake.

    Science.gov (United States)

    Tang, Xiangming; Gao, Guang; Zhu, Liping; Chao, Jianying; Qin, Boqiang

    2009-06-01

    Organic aggregates (OA) in aquatic ecosystems harbour diverse microbial communities. The colonization and growth of OA-attached bacteria are important processes in the degradation and transformation of the particles. The development of efficient and comparative DNA extraction methods is one of the most critical steps in the study of the composition and diversity of OA-attached bacterial communities. To evaluate whether different DNA extraction procedures affect the measurement of bacterial community composition, we compared four in situ lysis procedures using OA from three locations in a shallow eutrophic lake (Lake Taihu, China). The extracted DNA was analyzed using denaturing gradient gel electrophoresis profiles. We found that the choice of DNA extraction protocol had a significant influence on the fingerprints of the OA-attached bacterial community. This was shown not only in the number of bands but also in their relative representation of certain DNA bands. Using the bead-beating DNA extraction method in the presence of hexadecyltrimethylammonium bromide, we found that crude microbial DNA could be extracted efficiently from different OA types. This protocol is reproducible and gives very pure DNA of relatively high molecular mass. More importantly, the protocol provided more representative and informative data on the diversity of OA-attached bacterial communities.

  19. RAPID AND EFFICIENT METHOD FOR ENVIRONMENTAL DNA EXTRACTION AND PURIFICATION FROM SOIL

    Directory of Open Access Journals (Sweden)

    J. Hamedi

    2016-06-01

    Full Text Available Large proportion of microbial population in the world is unculturable. Extraction of total DNA from soil is usually a crucial step considering to the difficulties of study the uncultivable microorganisms. Humic acid is considered as the main inhibitory agent in the environmental DNA studies. Here, we introduced a rapid and efficient method for DNA extraction and purification from soil. Yield of DNA extraction by the presented method was 130 ng/µl. Three conventional methods of DNA extraction including liquid nitrogen incursion, bead beating and sonication were performed as control methods. Yield of DNA extraction by these methods were 110, 90 and 50 ng/µl, respectively. A rapid and efficient one step DNA purification method was introduced instead of hazardous conventional phenol-chloroform methods. Humic acid removal percentage by the introduced method was 95.8 % that is comparable with 97 % gained by the conventional gel extraction method and yield of DNA after purification was 84 % and 73 %, respectively. This study could be useful in molecular ecology and metagenomics study as a fast and reliable method.

  20. A salting out and resin procedure for extracting Schistosoma mansoni DNA from human urine samples

    Directory of Open Access Journals (Sweden)

    Rodrigues Nilton B

    2010-04-01

    Full Text Available Abstract Background In this paper a simple and cheap salting out and resin (InstaGene matrix® resin - BioRad DNA extraction method from urine for PCR assays is introduced. The DNA of the fluke Schistosoma mansoni was chosen as the target since schistosomiasis lacks a suitable diagnostic tool which is sensitive enough to detect low worm burden. It is well known that the PCR technique provides high sensitivity and specificity in detecting parasite DNA. Therefore it is of paramount importance to take advantage of its excellent performance by providing a simple to handle and reliable DNA extraction procedure, which permits the diagnosis of the disease in easily obtainable urine samples. Findings The description of the extraction procedure is given. This extraction procedure was tested for reproducibility and efficiency in artificially contaminated human urine samples. The reproducibility reached 100%, showing positive results in 5 assay repetitions of 5 tested samples each containing 20 ng DNA/5 ml. The efficiency of the extraction procedure was also evaluated in a serial dilution of the original 20 ng DNA/5 ml sample. Detectable DNA was extracted when it was at a concentration of 1.28 pg DNA/mL, revealing the high efficiency of this procedure. Conclusions This methodology represents a promising tool for schistosomiasis diagnosis utilizing a bio-molecular technique in urine samples which is now ready to be tested under field conditions and may be applicable to the diagnosis of other parasitic diseases.

  1. Microbial food safety: Potential of DNA extraction methods for use in diagnostic metagenomics.

    Science.gov (United States)

    Josefsen, Mathilde H; Andersen, Sandra C; Christensen, Julia; Hoorfar, Jeffrey

    2015-07-01

    The efficiency of ten widely applied DNA extraction protocols was evaluated for suitability for diagnostic metagenomics. The protocols were selected based on a thorough literature study. Chicken fecal samples inoculated with about 1×10(3) and 1×10(6) CFU/g Campylobacter jejuni were used as a model. The evaluation was performed based on total DNA yield measured by fluorometry, and quality and quantity of C. jejuni DNA measured by real-time PCR. There was up to a 25-fold variance between the lowest (NucliSens miniMAG, BIOMÉRIEUX) and highest (PowerLyzer PowerSoil DNA Isolation Kit, MO BIO Laboratories) yielding protocols. The PowerLyzer PowerSoil DNA Isolation Kit performed significantly better than all other protocols tested. Selected protocols were modified, i.e., extended heating and homogenization, resulting in increased yields of total DNA. For QIAamp Fast DNA Stool Mini Kit (Qiagen) a 7-fold increase in total DNA was observed following the protocol for human DNA analysis and including a 5 min heating step at 70°C. For the PowerLyzer PowerSoil and the PowerFecal DNA Isolation Kit (MO BIO Laboratories) the total DNA fold increase was 1.6 to 1.8 when including an extra 10 min of bead-vortexing. There was no correlation between the yield of total DNA and the amount of PCR-amplifiable DNA from C. jejuni. The protocols resulting in the highest yield of total DNA did not show correspondingly increased levels of C. jejuni DNA as determined by PCR. In conclusion, substantial variation in the efficiency of the protocols to extract DNA was observed. The highest DNA yield was obtained with the PowerLyzer PowerSoil DNA Isolation Kit, whereas the FastDNA SPIN Kit for Feces (MP Biomedicals) resulted in the highest amount of PCR-amplifiable C. jejuni DNA.

  2. Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil.

    Science.gov (United States)

    Wagner, Andreas O; Praeg, Nadine; Reitschuler, Christoph; Illmer, Paul

    2015-09-01

    Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol-chloroform-isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations.

  3. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    Science.gov (United States)

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

  4. A Rapid and Cost-Effective Method for DNA Extraction from Archival Herbarium Specimens.

    Science.gov (United States)

    Krinitsina, A A; Sizova, T V; Zaika, M A; Speranskaya, A S; Sukhorukov, A P

    2015-11-01

    Here we report a rapid and cost-effective method for the extraction of total DNA from herbarium specimens up to 50-90-year-old. The method takes about 2 h, uses AMPure XP magnetic beads diluted by PEG-8000- containing buffer, and does not require use of traditional volatile components like chloroform, phenol, and liquid nitrogen. It yields up to 4 µg of total nucleic acid with high purity from about 30 mg of dry material. The quality of the extracted DNA was tested by PCR amplification of 5S rRNA and rbcL genes (nuclear and chloroplast DNA markers) and compared against the traditional chloroform/isoamyl alcohol method. Our results demonstrate that the use of the magnetic beads is crucial for extraction of DNA suitable for subsequent PCR from herbarium samples due to the decreasing inhibitor concentrations, reducing short fragments of degraded DNA, and increasing median DNA fragment sizes.

  5. Genomic DNA from rat blood: A comparison of two extraction methods

    Directory of Open Access Journals (Sweden)

    Takić Miladinov, D.

    2016-09-01

    Full Text Available In this study, two methods for DNA extraction from fresh rat blood were compared. One is based on the use of cetyltrimethylammonium bromide (CTAB method, while the other one is well-known salting out method. Spectrophotometric analysis was employed to assess yield and purity of isolated DNA, while agarose gel electrophoresis was carried out to evaluate DNA integrity. The results have clearly demonstrated that the extraction method has significantly influenced the quantity and purity of isolated DNA. By using the CTAB method, a larger quantity of high-molecular weight DNA with good purity is obtained which, along with timeand cost-efficiency of the procedure, makes this method more suitable for the extraction of DNA from rat whole blood.

  6. High Purity DNA Extraction with a SPE Microfluidic Chip Using KI as the Binding Salt

    Institute of Scientific and Technical Information of China (English)

    Xing CHEN; Da Fu CUI; Chang Chun LIU

    2006-01-01

    Based on solid phase extraction method, a novel silicon-PDMS-glass microchip for high purity DNA extraction has been developed by using KI as the binding salt. The microfluidic chip fabricated by MEMS technology was composed of a silicon substrate with a coiled channel and a compounded PDMS-glass cover. With this microfluidic chip, the wall of the coiled channel was used as solid phase matrix for binding DNA and DNA was extracted by the fluxion of the binding buffer, washing buffer and elution buffer. KI as a substitute for guanidine, was used successfully as binding salt for purification DNA, obtaining higher purity of genomic DNA and about 13.9 ng DNA from 1 μL rat whole blood in 35 minutes.

  7. Complete sequence analysis of 18S rDNA based on genomic DNA extraction from individual Demodex mites (Acari: Demodicidae).

    Science.gov (United States)

    Zhao, Ya-E; Xu, Ji-Ru; Hu, Li; Wu, Li-Ping; Wang, Zheng-Hang

    2012-05-01

    The study for the first time attempted to accomplish 18S ribosomal DNA (rDNA) complete sequence amplification and analysis for three Demodex species (Demodex folliculorum, Demodex brevis and Demodex canis) based on gDNA extraction from individual mites. The mites were treated by DNA Release Additive and Hot Start II DNA Polymerase so as to promote mite disruption and increase PCR specificity. Determination of D. folliculorum gDNA showed that the gDNA yield reached the highest at 1 mite, tending to descend with the increase of mite number. The individual mite gDNA was successfully used for 18S rDNA fragment (about 900 bp) amplification examination. The alignments of 18S rDNA complete sequences of individual mite samples and those of pooled mite samples ( ≥ 1000mites/sample) showed over 97% identities for each species, indicating that the gDNA extracted from a single individual mite was as satisfactory as that from pooled mites for PCR amplification. Further pairwise sequence analyses showed that average divergence, genetic distance, transition/transversion or phylogenetic tree could not effectively identify the three Demodex species, largely due to the differentiation in the D. canis isolates. It can be concluded that the individual Demodex mite gDNA can satisfy the molecular study of Demodex. 18S rDNA complete sequence is suitable for interfamily identification in Cheyletoidea, but whether it is suitable for intrafamily identification cannot be confirmed until the ascertainment of the types of Demodex mites parasitizing in dogs.

  8. Human papillomavirus (HPV) in vulvar dysplasia and carcinoma in situ

    DEFF Research Database (Denmark)

    Junge, Jette; Poulsen, H; Horn, T;

    1995-01-01

    and 6 with HPV type 33) evenly distributed in all age groups and in all types of lesions (WHO and Toki et al. 1991). By ISH HPV was detected in 24/62 cases (39%) (21 with HPV type 16/18 and 3 with HPV type 31/33), nearly always in warty areas. All these cases were positive for the same virus type by PCR...... whereas no case of pure basaloid type was found. Various combinations of warty and basaloid types were shown in 52 cases and mixed forms in 7 cases. The results indicate that pure forms of warty and basaloid types probably do not exist. HPV DNA was detected by PCR in 51/58 cases (88%) (45 with HPV type 16....... No case revealed more than one type of HPV. HPV type 6, 11, 18, and 31 were not detected by PCR. The results indicate a correlation between HPV type 16 and 33 and dysplasia/carcinoma in situ in the vulva....

  9. DNA extract characterization process for microbial detection methods development and validation.

    Science.gov (United States)

    Olson, Nathan D; Morrow, Jayne B

    2012-12-03

    Quantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods. DNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/μL. Forty-seven extracts had concentrations greater than 17.5 ng/μL, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ μL), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/μL to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/μL and 0.25 ng/μL respectively. The qPCR LOQ varied by cell type (5.72 × 10-3 ng/μL for E. coli, 2.66 × 10-3 ng/μL, for B. cereus, 3.78 × 10-3 ng/μL for B. thailandensis, and 7.67 × 10-4 ng/μL for S. cerevisiae). A number of samples were below the UV spectroscopy (n = 27), flurometry (n = 15), and qPCR (n = 3) LOQ. The presented DNA extract

  10. Efficiency of genomic DNA extraction dependent on the size of magnetic nanoclusters

    Science.gov (United States)

    Cho, Hyun Ah; Hyun Min, Ji; Hua Wu, Jun; Woo Jang, Jin; Lim, Chae-Seung; Keun Kim, Young

    2014-05-01

    We report the efficiency of genomic DNA extraction as a function of particle size and quantity. For DNA extraction, we synthesized magnetic nanoclusters of various sizes and coated the surface of these magnetic nanoclusters with meso-2,3-dimercaptosuccinic acid. We showed that the nanoclusters had a tight particle size distribution and high crystallinity. Furthermore, we observed that the three types of magnetic nanoclusters studied exhibited ferrimagnetic behavior and that larger nanoclusters showed larger saturation magnetization values. The resultant efficiency of DNA extraction is inversely proportional to particle size in the range of nanoclusters tested, due to the fact that the surface-to-volume ratio decreases as particle size increases.

  11. Evaluating DNA Extraction Methods for Community Profiling of Pig Hindgut Microbial Community.

    Science.gov (United States)

    Lu, Yang; Hugenholtz, Philip; Batstone, Damien John

    2015-01-01

    Recovery of high quality PCR-amplifiable DNA has been the general minimal requirement for DNA extraction methods for bulk molecular analysis. However, modern high through-put community profiling technologies are more sensitive to representativeness and reproducibility of DNA extraction method. Here, we assess the impact of three DNA extraction methods (with different levels of extraction harshness) for assessing hindgut microbiomes from pigs fed with different diets (with different physical properties). DNA extraction from each sample was performed in three technical replicates for each extraction method and sequenced by 16S rRNA amplicon sequencing. Host was the primary driver of molecular sequencing outcomes, particularly on samples analysed by wheat based diets, but higher variability, with one failed extraction occurred on samples from a barley fed pig. Based on these results, an effective method will enable reproducible and quality outcomes on a range of samples, whereas an ineffective method will fail to generate extract, but host (rather than extraction method) remains the primary factor.

  12. DNA extraction of ancient animal hard tissue samples via adsorption to silica particles.

    Science.gov (United States)

    Rohland, Nadin

    2012-01-01

    A large number of subfossil and more recent skeletal remains, many of which are stored in museums and private collections, are potentially accessible for DNA sequence analysis. In order to extract the small amount of DNA preserved in these specimens, an efficient DNA release and purification method is required. In this chapter, I describe an efficient and straightforward purification and concentration method that uses DNA adsorption to a solid surface of silica particles. Comparative analysis of extraction methods has shown that this method works reliably for ancient as well as younger, museum-preserved specimens.

  13. An evaluation of the performance of five extraction methods: Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™.

    Science.gov (United States)

    Ip, Stephen C Y; Lin, Sze-Wah; Lai, Kam-Ming

    2015-05-01

    DNA left at a crime scene was often limited in amount and far from pristine. To maximize the chance of recovering as much information as possible from such compromised samples, an appropriate extraction method using the available technologies needs to be devised. In this study, we used human blood, buffy coat and a total of 76 simulated touch DNA samples to test the effectiveness of the following five common DNA extraction methods, namely, Chelex® 100, QIAamp® DNA Blood Mini Kit, QIAamp® DNA Investigator Kit, QIAsymphony® DNA Investigator® Kit and DNA IQ™ system, in the recovery of such DNA. We demonstrated that the QIAamp® and QIAsymphony® DNA Investigator® Kits, and the DNA IQ™ system, exhibited a better effectiveness in DNA recovery amongst these methods and yielded extracts with higher success rate in subsequent DNA profiling. These extracts also generated profiles with better intra-colour signal balance. The findings in this work allowed us to propose an extraction approach as follows: 1) casework samples shall be extracted with the QIAamp®/QIAsymphony® DNA Investigator® Kits or the DNA IQ™ system, viz., QIAsymphony® DNA Investigator® Kit and DNA IQ™, due to their higher throughput, are for the touched DNA evidence from the volume crime, while QIAamp® DNA Investigator Kit is preferable for challenging bloodstain samples; and 2) control samples, such as buccal swab, with known identity can be extracted with the Chelex, due to their cheaper cost per sample. Copyright © 2015 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

  14. Primary cervical cancer screening with HPV testing compared with liquid-based cytology: results of round 1 of a randomised controlled trial -- the HPV FOCAL Study.

    Science.gov (United States)

    Ogilvie, G S; Krajden, M; van Niekerk, D J; Martin, R E; Ehlen, T G; Ceballos, K; Smith, L W; Kan, L; Cook, D A; Peacock, S; Stuart, G C E; Franco, E L; Coldman, A J

    2012-12-04

    Round 1 data of human papillomavirus (HPV) FOCAL, a three-arm, randomised trial, which aims to establish the efficacy of HPV DNA testing as a primary screen for cervical cancer, are presented. The three arms are: Control arm - liquid based cytology with atypical squamous cells of unknown significance (ASC-US) triage with hrHPV testing; Intervention Arm - hrHPV at entry with liquid-based cytology (LBC) triage of hrHPV positives, with exit screen at 4 years; Safety check arm - hrHPV at entry with LBC triage of hrHPV positives with exit screen at 2 years. A total of 6154 women were randomised to the control arm and 12 494 to the HPV arms (intervention and safety check). In the HPV arm, the baseline cervical intraepithelial neoplasia (CIN)2+ and CIN3+ rate was 9.2/1000 (95%CI; 7.4, 10.9) and 4.8/1000 (95%CI; 3.6, 6.1), which increased to 16.1/1000 (95%CI 13.2, 18.9) for CIN2+ and to 8.0/1000 (95%CI; 5.9, 10.0) for CIN3+ after subsequent screening of HPV-DNA-positive/cytology-negative women. Detection rate in the control arm remained unchanged after subsequent screening of ASC-US-positive/hrHPV DNA-negative women at 11.0/1000 for CIN2+ and 5.0/1000 for CIN3+. After subsequent screening of women who were either hrHPV positive/cytology negative or ASC-US positive/HPV negative, women randomised to the HPV arms had increased CIN2+ detection compared with women randomised to the cytology arm.

  15. Primary cervical cancer screening with HPV testing compared with liquid-based cytology: results of round 1 of a randomised controlled trial – the HPV FOCAL Study

    Science.gov (United States)

    Ogilvie, G S; Krajden, M; van Niekerk, D J; Martin, R E; Ehlen, T G; Ceballos, K; Smith, L W; Kan, L; Cook, D A; Peacock, S; Stuart, G C E; Franco, E L; Coldman, A J

    2012-01-01

    Background: Round 1 data of human papillomavirus (HPV) FOCAL, a three-arm, randomised trial, which aims to establish the efficacy of HPV DNA testing as a primary screen for cervical cancer, are presented. Methods: The three arms are: Control arm – liquid based cytology with atypical squamous cells of unknown significance (ASC-US) triage with hrHPV testing; Intervention Arm – hrHPV at entry with liquid-based cytology (LBC) triage of hrHPV positives, with exit screen at 4 years; Safety check arm – hrHPV at entry with LBC triage of hrHPV positives with exit screen at 2 years. Results: A total of 6154 women were randomised to the control arm and 12 494 to the HPV arms (intervention and safety check). In the HPV arm, the baseline cervical intraepithelial neoplasia (CIN)2+ and CIN3+ rate was 9.2/1000 (95%CI; 7.4, 10.9) and 4.8/1000 (95%CI; 3.6, 6.1), which increased to 16.1/1000 (95%CI 13.2, 18.9) for CIN2+ and to 8.0/1000 (95%CI; 5.9, 10.0) for CIN3+ after subsequent screening of HPV-DNA-positive/cytology-negative women. Detection rate in the control arm remained unchanged after subsequent screening of ASC-US-positive/hrHPV DNA-negative women at 11.0/1000 for CIN2+ and 5.0/1000 for CIN3+. Conclusion: After subsequent screening of women who were either hrHPV positive/cytology negative or ASC-US positive/HPV negative, women randomised to the HPV arms had increased CIN2+ detection compared with women randomised to the cytology arm. PMID:23169286

  16. Elimination of bioweapons agents from forensic samples during extraction of human DNA.

    Science.gov (United States)

    Timbers, Jason; Wilkinson, Della; Hause, Christine C; Smith, Myron L; Zaidi, Mohsin A; Laframboise, Denis; Wright, Kathryn E

    2014-11-01

    Collection of DNA for genetic profiling is a powerful means for the identification of individuals responsible for crimes and terrorist acts. Biologic hazards, such as bacteria, endospores, toxins, and viruses, could contaminate sites of terrorist activities and thus could be present in samples collected for profiling. The fate of these hazards during DNA isolation has not been thoroughly examined. Our goals were to determine whether the DNA extraction process used by the Royal Canadian Mounted Police eliminates or neutralizes these agents and if not, to establish methods that render samples safe without compromising the human DNA. Our results show that bacteria, viruses, and toxins were reduced to undetectable levels during DNA extraction, but endospores remained viable. Filtration of samples after DNA isolation eliminated viable spores from the samples but left DNA intact. We also demonstrated that contamination of samples with some bacteria, endospores, and toxins for longer than 1 h compromised the ability to complete genetic profiling.

  17. Sample pretreatment microfluidic chip for DNA extraction from rat peripheral blood

    Institute of Scientific and Technical Information of China (English)

    CHEN Xing; CUI Dafu; LIU Changchun; LI Hui; ZHAO Weixing

    2007-01-01

    A sample pretreatment microfluidic chip was described based on the principle of solid phase extraction and micro electro mechanical system technology.Oxidized porous silicon with the large surface area as the solid phase matrix for absorption of DNA from a biological sample can greatly improve the DNA yield.The factors that could affect the DNA yield were analyzed and the preparation technology and the experiment procedure were improved.The DNA purification process from the rat peripheral blood can be achieved and the DNA yield is 24 ng/(μL whole blood),which can reach the level of the commercial DNA purification kits.Furthermore,the DNA extracted from the whole blood can be amplified by polymerase chain reaction,which can achieve a high efficiency of the amplification.

  18. A randomized controlled trial of Human Papillomavirus (HPV testing for cervical cancer screening: trial design and preliminary results (HPV FOCAL Trial

    Directory of Open Access Journals (Sweden)

    Smith Laurie W

    2010-03-01

    Full Text Available Abstract Background In the HPV FOCAL trial, we will establish the efficacy of hr-HPV DNA testing as a stand-alone screening test followed by liquid based cytology (LBC triage of hr-HPV-positive women compared to LBC followed by hr-HPV triage with ≥ CIN3 as the outcome. Methods/Design HPV-FOCAL is a randomized, controlled, three-armed study over a four year period conducted in British Columbia. It will recruit 33,000 women aged 25-65 through the province's population based cervical cancer screening program. Control arm: LBC at entry and two years, and combined LBC and hr-HPV at four years among those with initial negative results and hr-HPV triage of ASCUS cases; Two Year Safety Check arm: hr-HPV at entry and LBC at two years in those with initial negative results with LBC triage of hr-HPV positives; Four Year Intervention Arm: hr-HPV at entry and combined hr-HPV and LBC at four years among those with initial negative results with LBC triage of hr-HPV positive cases Discussion To date, 6150 participants have a completed sample and epidemiologic questionnaire. Of the 2019 women enrolled in the control arm, 1908 (94.5% were cytology negative. Women aged 25-29 had the highest rates of HSIL (1.4%. In the safety arm 92.2% of women were hr-HPV negative, with the highest rate of hr-HPV positivity found in 25-29 year old women (23.5%. Similar results were obtained in the intervention arm HPV FOCAL is the first randomized trial in North America to examine hr-HPV testing as the primary screen for cervical cancer within a population-based cervical cancer screening program. Trial Registration International Standard Randomised Controlled Trial Number Register, ISRCTN79347302

  19. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2017-08-29

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  20. Characterisation of human papillomavirus (HPV genotypes in the Azorean population, Terceira island

    Directory of Open Access Journals (Sweden)

    Teixeira Fernando

    2008-04-01

    Full Text Available Abstract Background Human papillomavirus detection is very important for the evaluation of prevention strategies in cervical cancer. In the Azorean population, the virus prevalence has never been studied, and there is no data available to preview a successful outcome with HPV vaccination. In this article, our objective is to characterise the HPV genotypes in Terceira Island, contributing for the epidemiological knowledge on the virus infection. Results Cervical samples were collected from 289 women aged 16–81 in the Gynaecological Outpatient Clinic of the Hospital de Santo Espírito de Angra do Heroísmo (HSEAH. HPV DNA was amplified by Polymerase Chain Reaction using the general consensus primers PGMYO9/PGMY11. Commercially available Papillomavirus Clinical Arrays® kits (Genomica were used to perform HPV genotyping. 30 women were HPV positive, with a median age of 41 years old. Our results show that the overall HPV prevalence was 10.49%. Seventeen genotypes were identified, including 58.82% high risk, 17.65% low risk and 23.53% undetermined risk. Conclusion Unlike other epidemiological studies, HPV31 was the most frequent type (26.67% in Terceira Island, followed by HPV16 (10.00%, HPV51, HPV53, HPV70 and HPV82 (6.67%. Further studies are needed to investigate if the HPV types found in our population are associated with the risk of progression to high-grade squamous intraepithelial lesions or cervical cancer.

  1. Characterization of two novel gammapapillomaviruses, HPV179 and HPV184, isolated from common warts of a renal-transplant recipient.

    Science.gov (United States)

    Hošnjak, Lea; Kocjan, Boštjan J; Pirš, Branko; Seme, Katja; Poljak, Mario

    2015-01-01

    Gammapapillomavirus (Gamma-PV) is a diverse and rapidly expanding PV-genus, currently consisting of 76 fully characterized human papillomavirus (HPV) types. In this study, DNA genomes of two novel HPV types, HPV179 and HPV184, obtained from two distinct facial verrucae vulgares specimens of a 64 year-old renal-transplant recipient, were fully cloned, sequenced and characterized. HPV179 and HPV184 genomes comprise 7,228-bp and 7,324-bp, respectively, and contain four early (E1, E2, E6 and E7) and two late genes (L1 and L2); the non-coding region is typically positioned between L1 and E6 genes. Phylogenetic analysis of the L1 nucleotide sequence placed both novel types within the Gamma-PV genus: HPV179 was classified as a novel member of species Gamma-15, additionally containing HPV135 and HPV146, while HPV184 was classified as a single member of a novel species Gamma-25. HPV179 and HPV184 type-specific quantitative real-time PCRs were further developed and used in combination with human beta-globin gene quantitative real-time PCR to determine the prevalence and viral load of the novel types in the patient's facial warts and several follow-up skin specimens, and in a representative collection, a total of 569 samples, of HPV-associated benign and malignant neoplasms, hair follicles and anal and oral mucosa specimens obtained from immunocompetent individuals. HPV179 and HPV184 viral loads in patients' facial warts were estimated to be 2,463 and 3,200 genome copies per single cell, respectively, suggesting their active role in the development of common warts in organ-transplant recipients. In addition, in this particular patient, both novel types had established a persistent infection of the skin for more than four years. Among immunocompetent individuals, HPV179 was further detected in low-copy numbers in a few skin specimens, indicating its cutaneous tissue tropism, while HPV184 was further detected in low-copy numbers in one mucosal and a few skin specimens

  2. DNA extraction protocol for biological ingredient analysis of Liuwei Dihuang Wan.

    Science.gov (United States)

    Cheng, Xinwei; Chen, Xiaohua; Su, Xiaoquan; Zhao, Huanxin; Han, Maozhen; Bo, Cunpei; Xu, Jian; Bai, Hong; Ning, Kang

    2014-06-01

    Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological contaminants. For biological ingredient analysis, the efficiency of DNA extraction is an important factor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB) and three commonly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out. The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3-4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations. Copyright © 2014. Production and hosting by Elsevier Ltd.

  3. DNA Extraction Protocol for Biological Ingredient Analysis of Liuwei Dihuang Wan

    Institute of Scientific and Technical Information of China (English)

    Xinwei Cheng; Xiaohua Chen; Xiaoquan Su; Huanxin Zhao; Maozhen Han; Cunpei Bo; Jian Xu; Hong Bai; Kang Ning

    2014-01-01

    Traditional Chinese medicine (TCM) preparations are widely used for healthcare and clinical practice. So far, the methods commonly used for quality evaluation of TCM preparations mainly focused on chemical ingredients. The biological ingredient analysis of TCM preparations is also important because TCM preparations usually contain both plant and animal ingredients, which often include some mis-identified herbal materials, adulterants or even some biological con-taminants. For biological ingredient analysis, the efficiency of DNA extraction is an important fac-tor which might affect the accuracy and reliability of identification. The component complexity in TCM preparations is high, and DNA might be destroyed or degraded in different degrees after a series of processing procedures. Therefore, it is necessary to establish an effective protocol for DNA extraction from TCM preparations. In this study, we chose a classical TCM preparation, Liuwei Dihuang Wan (LDW), as an example to develop a TCM-specific DNA extraction method. An optimized cetyl trimethyl ammonium bromide (CTAB) method (TCM-CTAB) and three com-monly-used extraction kits were tested for extraction of DNA from LDW samples. Experimental results indicated that DNA with the highest purity and concentration was obtained by using TCM-CTAB. To further evaluate the different extraction methods, amplification of the second internal transcribed spacer (ITS2) and the chloroplast genome trnL intron was carried out.The results have shown that PCR amplification was successful only with template of DNA extracted by using TCM-CTAB. Moreover, we performed high-throughput 454 sequencing using DNA extracted by TCM-CTAB. Data analysis showed that 3-4 out of 6 prescribed species were detected from LDW samples, while up to 5 contaminating species were detected, suggesting TCM-CTAB method could facilitate follow-up DNA-based examination of TCM preparations.

  4. DNA extraction methods for detecting genetically modified foods: A comparative study.

    Science.gov (United States)

    Elsanhoty, Rafaat M; Ramadan, Mohamed Fawzy; Jany, Klaus Dieter

    2011-06-15

    The work presented in this manuscript was achieved to compare six different methods for extracting DNA from raw maize and its derived products. The methods that gave higher yield and quality of DNA were chosen to detect the genetic modification in the samples collected from the Egyptian market. The different methods used were evaluated for extracting DNA from maize kernels (without treatment), maize flour (mechanical treatment), canned maize (sweet corn), frozen maize (sweet corn), maize starch, extruded maize, popcorn, corn flacks, maize snacks, and bread made from corn flour (mechanical and thermal treatments). The quality and quantity of the DNA extracted from the standards, containing known percentages of GMO material and from the different food products were evaluated. For qualitative detection of the GMO varieties in foods, the GMOScreen 35S/NOS test kit was used, to screen the genetic modification in the samples. The positive samples for the 35S promoter and/or the NOS terminator were identified by the standard methods adopted by EU. All of the used methods extracted yielded good DNA quality. However, we noted that the purest DNA extract were obtained using the DNA extraction kit (Roche) and this generally was the best method for extracting DNA from most of the maize-derived foods. We have noted that the yield of DNA extracted from maize-derived foods was generally lower in the processed products. The results indicated that 17 samples were positive for the presence of 35S promoter, while 34% from the samples were positive for the genetically modified maize line Bt-176.

  5. Comparing DNA extraction methods for analysis of botanical materials found in anti-diabetic supplements.

    Science.gov (United States)

    Llongueras, Jose P; Nair, Saraswathy; Salas-Leiva, Dayana; Schwarzbach, Andrea E

    2013-03-01

    A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A(260)/A(280) between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A(260)/A(280) ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A(260)/A(280) measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin(®) plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.

  6. Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

    Science.gov (United States)

    Nagai, Momoko; Minegishi, Katsura; Komada, Munekazu; Tsuchiya, Maiko; Kameda, Tomomi; Yamada, Shigehito

    2016-05-01

    The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies. © 2015 Japanese Teratology Society.

  7. DNA extraction methods and multiple sampling to improve molecular diagnosis of Sarcocystis spp. in cattle hearts.

    Science.gov (United States)

    Bräunig, Patrícia; Portella, Luiza Pires; Cezar, Alfredo Skrebsky; Libardoni, Felipe; Sangioni, Luis Antonio; Vogel, Fernanda Silveira Flores; Gonçalves, Paulo Bayard Dias

    2016-10-01

    Molecular detection of Sarcocystis spp. in tissue samples can be useful for experimental and diagnostic purposes. However, the parasite spreads unevenly through tissues, forming tissue cysts, and the cystic wall is an obstacle in DNA extraction protocols. Therefore, adequate sampling and effective disruption of the cysts are essential to improve the accuracy of DNA detection by PCR. The aims of this study were to evaluate the suitability of four protocols for DNA extraction from cysts of Sarcocystis spp. present in bovine myocardium samples or after their harvest in phosphate-buffered saline (PBS) solution as well as determine the effects of single or multiple sampling on the accuracy of molecular diagnosis of sarcocystosis in cattle hearts. Cysts and myocardium samples from nine bovine hearts were randomly distributed to four DNA extraction protocols: kit, kit with modification, DNAzol, and cetyl-trimethyl ammonium bromide (CTAB). Samples were submitted to DNA extraction and PCR as replicates of each heart (simplicate, duplicate, and triplicate), and the probability of a true positive diagnostic was calculated. Among the protocols tested, the kit with modification was determined to be the most suitable for DNA extraction from cysts in PBS solution (92.6 % of DNA detection by PCR); DNAzol resulted in higher DNA detection frequency from bovine myocardium samples (48.1 %). Multiple sampling improved the molecular diagnosis of Sarcocystis spp. infection in cattle hearts, increasing at 22.2 % the rate of true positive diagnostic.

  8. Standardization of DNA extraction from sand flies: Application to genotyping by next generation sequencing.

    Science.gov (United States)

    Casaril, Aline Etelvina; de Oliveira, Liliane Prado; Alonso, Diego Peres; de Oliveira, Everton Falcão; Gomes Barrios, Suellem Petilim; de Oliveira Moura Infran, Jucelei; Fernandes, Wagner de Souza; Oshiro, Elisa Teruya; Ferreira, Alda Maria Teixeira; Ribolla, Paulo Eduardo Martins; de Oliveira, Alessandra Gutierrez

    2017-06-01

    Standardization of the methods for extraction of DNA from sand flies is essential for obtaining high efficiency during subsequent molecular analyses, such as the new sequencing methods. Information obtained using these methods may contribute substantially to taxonomic, evolutionary, and eco-epidemiological studies. The aim of the present study was to standardize and compare two methods for the extraction of genomic DNA from sand flies for obtaining DNA in sufficient quantities for next-generation sequencing. Sand flies were collected from the municipalities of Campo Grande, Camapuã, Corumbá and Miranda, state of Mato Grosso do Sul, Brazil. Three protocols using a silica column-based commercial kit (ReliaPrep™ Blood gDNA Miniprep System kit, Promega(®)), and three protocols based on the classical phenol-chloroform extraction method (Uliana et al., 1991), were compared with respect to the yield and quality of the extracted DNA. DNA was quantified using a Qubit 2.0 fluorometer. The presence of sand fly DNA was confirmed by PCR amplification of the IVS6 region (constitutive gene), followed by electrophoresis on a 1.5% agarose gel. A total of 144 male specimens were analyzed, 72 per method. Significant differences were observed between the two methods tested. Protocols 2 and 3 of phenol-chloroform extraction presented significantly better performance than all commercial kit extraction protocols tested. For phenol-chloroform extraction, protocol 3 presented significantly better performance than protocols 1 and 2. The IVS6 region was detected in 70 of 72 (97.22%) samples extracted with phenol, including all samples for protocols 2 and 3. This is the first study on the standardization of methods for the extraction of DNA from sand flies for application to next-generation sequencing, which is a promising tool for entomological and molecular studies of sand flies. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. An Efficient DNA Extraction Method for Lactobacillus casei, a Difficult-to-Lyse Bacterium

    Directory of Open Access Journals (Sweden)

    Alimolaei

    2016-02-01

    Full Text Available Background There are several protocols to extract DNA from Lactobacillus spp. In the case of L. casei it is harder because of its especial and thick cell wall. Objectives In this study, nine DNA extraction protocols (by lysozyme treatment were evaluated and compared in two categories (traditional and kit-based protocols and an improved method was presented. Materials and Methods DNA quantity and quality was determined by spectrophotometry, agarose gel electrophoresis and polymerase chain reaction (PCR. Results The results revealed that the yield of extracted DNA differed by each protocol (5.8 - 17.1 μg/100 μL, but provided appropriate DNA for PCR amplification. The modified protocol offered the best total DNA extraction method when both quality (DNA purity; 1.54 μg and quantity (DNA yield; 17.1 μg were considered. Conclusions We suggest this protocol for effective and inexpensive DNA isolation from L. casei for downstream biological processes such as PCR.

  10. Increasing DNA extraction yield from saliva stains with a modified Chelex method.

    Science.gov (United States)

    Sweet, D; Lorente, M; Valenzuela, A; Lorente, J A; Alvarez, J C

    1996-12-27

    Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex extraction were compared to a modified Chelex method developed by the authors. Modifications include pre-extraction preparation with proteinase K and incubations at 56 degrees C and 100 degrees C plus microconcentration of the solution. Quantification results using the classical Chelex extraction method showed that 31.9 +/- 4.22% of the deposited DNA was recovered, but using the modified Chelex extraction method DNA recovery was increased to 47.7 +/- 6.90%. The quantity and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

  11. Evaluation protocols for the extraction of genomic DNA from Bovine blood

    Directory of Open Access Journals (Sweden)

    Crispim Bruno do Amaral

    2016-08-01

    Full Text Available Basic studies on DNA extraction techniques are very important for the success of scientific papers in the field of molecular biology. The extraction and purification of nucleic acids are critical steps for establishing further genetic analysis. The objective of this study was to evaluate the efficacy of different protocols for DNA extraction by determining the quantity and quality of extracted genetic material and the possibility of amplification by PCR. We did DNA extraction and PCR of ten bovine blood samples. The test results obtained by spectrophotometry indicated that the quantity and quality of genomic DNA were considered satisfactory in all protocols for PCR. However, there was a statistically significant difference between the parameters measured, both in quantity and in quality (p <0.01. The extraction protocol using whole blood was more efficient in terms of time and quality; there was no degradation in all processes of extraction. It was also demonstrated that the possibility of amplification of the region of exon 2 of the leptin gene in extracted DNA exists.

  12. Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections

    Science.gov (United States)

    He, Hui; Li, Rongqun; Chen, Yi; Pan, Ping; Tong, Wenjuan; Dong, Xueyan; Chen, Yueming; Yu, Daojun

    2017-01-01

    Current extraction methods often extract DNA and RNA separately, and few methods are capable of co-extracting DNA and RNA from sputum. We established a nucleic acid co-extraction method from sputum based on magnetic beads and optimized the method by evaluating influencing factors, such as the guanidinium thiocyanate (GTC) and dithiothreitol (DTT) concentrations, magnetic bead amount, incubation temperature, lysis buffer pH and RNA carrier type. The feasibility of the simultaneous nucleic acid co-extraction method was evaluated by amplifying DNA and RNA viruses from a single clinical specimen with a multiplex RT-qPCR method. Both DNA and RNA were most efficiently extracted when the GTC and DTT concentrations were 2.0 M and 80 mM, respectively, 20 μl magnetic beads were added, the incubation temperature was 80 °C, the pH was 8 or 9, and RNA carrier A was used. Therefore, we established a simple method to extract nucleic acids from two important respiratory viruses compared with other commercial kits. This magnetic beads-based co-extraction method for sputum followed by a multiplex RT-qPCR can rapidly and precisely detect DNA and RNA viruses from a single clinical specimen and has many advantages, such as decreased time, low cost, and a lack of harmful chemicals. PMID:28332631

  13. Genomic DNA extraction from medicinal plants available in Malaysia using a TriOmic(TM) improved extraction kit.

    Science.gov (United States)

    Mohd-Hairul, A R; Sade, A B; Yiap, B C; Raha, A R

    2011-11-08

    DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete.

  14. Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR

    Directory of Open Access Journals (Sweden)

    André Moura

    2016-07-01

    Full Text Available ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evaluated when combined with two real time PCRs. The DNeasy Blood & Tissue Kit from QIAGEN showed better performance and sensitivity followed by the DNA Mini Kit RBC and FTA Elute Micro Card. Results suggested that, even when the analytical sensitivity of the qPCR is very high, the extraction method can influence the diagnostic sensitivity.

  15. Simplified extraction of good quality genomic DNA from a variety of plant materials: 1

    National Research Council Canada - National Science Library

    Vijay Kumari; Anshu Bansal; Raghavendra Aminedi; Dhakshi Taneja; Niranjan Das

    2012-01-01

      Depending on the nature and complexity of plant material, proper method needs to be employed for extraction of genomic DNA, along with its performance evaluation by different molecular techniques...

  16. Does DNA extraction affect the physical and chemical composition of historical cod (Gadus morhua) otoliths?

    DEFF Research Database (Denmark)

    Therkildsen, Nina Overgaard; Eg Nielsen, Einar; Hüssy, Karin

    2010-01-01

    conflicting interests regarding how the limited and irreplaceable otolith collections should be used. To resolve this, it is important to find out whether DNA extraction damages otoliths such that they can no longer be used for other research purposes or whether individual otoliths can be used in multiple...... applications. We examined the effects of three different DNA extraction methods on the elemental composition, the morphology, and the clarity of annual growth increments for successful age estimation of Atlantic cod (Gadus morhua) otoliths that had been archived for 0–31 years. The three extraction methods...... yielded DNA of comparable quality, and none of the methods caused major damage to the otoliths. Of the element concentrations measured, only Mg and Rb showed considerable changes resulting from DNA extraction. The physical properties of the otolith (morphology and clarity of annual growth increments) were...

  17. A protocol for high-quality genomic DNA extraction from legumes

    National Research Council Canada - National Science Library

    Agbagwa, I O; Datta, S; Patil, P G; Singh, P; Nadarajan, N

    2012-01-01

    ... in less researched crops in laboratories in developing countries. We modified and optimized the existing CTAB method for plant genomic DNA extraction by avoiding liquid nitrogen usage and lyophilization...

  18. A simple method of DNA extraction from coffee seeds suitable for ...

    African Journals Online (AJOL)

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    2008-02-19