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Sample records for mlra gene-directed taqman

  1. Heterologous expression of mlrA in a photoautotrophic host - Engineering cyanobacteria to degrade microcystins.

    Science.gov (United States)

    Dexter, Jason; Dziga, Dariusz; Lv, Jing; Zhu, Junqi; Strzalka, Wojciech; Maksylewicz, Anna; Maroszek, Magdalena; Marek, Sylwia; Fu, Pengcheng

    2018-06-01

    In this report, we establish proof-of-principle demonstrating for the first time genetic engineering of a photoautotrophic microorganism for bioremediation of naturally occurring cyanotoxins. In model cyanobacterium Synechocystis sp. PCC 6803 we have heterologously expressed Sphingopyxis sp. USTB-05 microcystinase (MlrA) bearing a 23 amino acid N-terminus secretion peptide from native Synechocystis sp. PCC 6803 PilA (sll1694). The resultant whole cell biocatalyst displayed about 3 times higher activity against microcystin-LR compared to a native MlrA host (Sphingomonas sp. ACM 3962), normalized for optical density. In addition, MlrA activity was found to be almost entirely located in the cyanobacterial cytosolic fraction, despite the presence of the secretion tag, with crude cellular extracts showing MlrA activity comparable to extracts from MlrA expressing E. coli. Furthermore, despite approximately 9.4-fold higher initial MlrA activity of a whole cell E. coli biocatalyst, utilization of a photoautotrophic chassis resulted in prolonged stability of MlrA activity when cultured under semi-natural conditions (using lake water), with the heterologous MlrA biocatalytic activity of the E. coli culture disappearing after 4 days, while the cyanobacterial host displayed activity (3% of initial activity) after 9 days. In addition, the cyanobacterial cell density was maintained over the duration of this experiment while the cell density of the E. coli culture rapidly declined. Lastly, failure to establish a stable cyanobacterial isolate expressing native MlrA (without the N-terminus tag) via the strong cpcB560 promoter draws attention to the use of peptide tags to positively modulate expression of potentially toxic proteins. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Stx1 prophage excision in Escherichia coli strain PA20 confers strong curli and biofilm formation by restoring native mlrA

    Science.gov (United States)

    Prophage insertions in Escherichia coli O157:H7 mlrA contribute to the low expression of curli fimbriae and biofilm observed in many clinical isolates. Varying levels of CsgD-dependent curli/biofilm expression are restored to strains bearing prophage insertions in mlrA by mutation of regulatory gene...

  3. Bystander or No Bystander for Gene Directed Enzyme Prodrug Therapy

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    Adam V. Patterson

    2009-11-01

    Full Text Available Gene directed enzyme prodrug therapy (GDEPT of cancer aims to improve the selectivity of chemotherapy by gene transfer, thus enabling target cells to convert nontoxic prodrugs to cytotoxic drugs. A zone of cell kill around gene-modified cells due to transfer of toxic metabolites, known as the bystander effect, leads to tumour regression. Here we discuss the implications of either striving for a strong bystander effect to overcome poor gene transfer, or avoiding the bystander effect to reduce potential systemic effects, with the aid of three successful GDEPT systems. This review concentrates on bystander effects and drug development with regard to these enzyme prodrug combinations, namely herpes simplex virus thymidine kinase (HSV-TK with ganciclovir (GCV, cytosine deaminase (CD from bacteria or yeast with 5-fluorocytodine (5-FC, and bacterial nitroreductase (NfsB with 5-(azaridin-1-yl-2,4-dinitrobenzamide (CB1954, and their respective derivatives.

  4. Interim Report on Multiple Sequence Alignments and TaqMan Signature Mapping to Phylogenetic Trees

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, S; Jaing, C

    2012-03-27

    The goal of this project is to develop forensic genotyping assays for select agent viruses, addressing a significant capability gap for the viral bioforensics and law enforcement community. We used a multipronged approach combining bioinformatics analysis, PCR-enriched samples, microarrays and TaqMan assays to develop high resolution and cost effective genotyping methods for strain level forensic discrimination of viruses. We have leveraged substantial experience and efficiency gained through year 1 on software development, SNP discovery, TaqMan signature design and phylogenetic signature mapping to scale up the development of forensics signatures in year 2. In this report, we have summarized the Taqman signature development for South American hemorrhagic fever viruses, tick-borne encephalitis viruses and henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus and Japanese encephalitis virus.

  5. Real-time TaqMan polymerase chain reaction to quantify the effects ...

    African Journals Online (AJOL)

    TaqMan polymerase chain reaction was developed to quantify the number of Bifidobacterium. We used this assay to detect genomic DNA of Bifidobacterium in the intestinal tract digesta of piglets, including duodenum, jejunum, ileum, cecum and colon. Our results indicated that, developed new real-time quantitative PCR ...

  6. Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species

    NARCIS (Netherlands)

    Wolf, van der J.M.; Haas, de B.H.; Hoof, van R.A.; Haan, de E.G.; Bovenkamp, van den G.W.

    2014-01-01

    TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic

  7. T.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory

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    Albion Tim

    2005-10-01

    Full Text Available Abstract Background Single Nucleotide Polymorphism (SNP genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS need tools to organize the data flow. Results We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements. Conclusion We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories whithout a more sophisticated and expensive system, such as a LIMS.

  8. T.I.M.S: TaqMan Information Management System, tools to organize data flow in a genotyping laboratory

    Science.gov (United States)

    Monnier, Stéphanie; Cox, David G; Albion, Tim; Canzian, Federico

    2005-01-01

    Background Single Nucleotide Polymorphism (SNP) genotyping is a major activity in biomedical research. The Taqman technology is one of the most commonly used approaches. It produces large amounts of data that are difficult to process by hand. Laboratories not equipped with a Laboratory Information Management System (LIMS) need tools to organize the data flow. Results We propose a package of Visual Basic programs focused on sample management and on the parsing of input and output TaqMan files. The code is written in Visual Basic, embedded in the Microsoft Office package, and it allows anyone to have access to those tools, without any programming skills and with basic computer requirements. Conclusion We have created useful tools focused on management of TaqMan genotyping data, a critical issue in genotyping laboratories whithout a more sophisticated and expensive system, such as a LIMS. PMID:16221298

  9. A Laboratory-Developed TaqMan Array Card for Simultaneous Detection of 19 Enteropathogens

    Science.gov (United States)

    Liu, Jie; Gratz, Jean; Amour, Caroline; Kibiki, Gibson; Becker, Stephen; Janaki, Lalitha; Verweij, Jaco J.; Taniuchi, Mami; Sobuz, Shihab U.; Haque, Rashidul; Haverstick, Doris M.

    2013-01-01

    The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and

  10. Identification of field caught Anopheles gambiae s.s. and Anopheles arabiensis by TaqMan single nucleotide polymorphism genotyping

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    Bayoh Nabie M

    2007-02-01

    Full Text Available Abstract Background Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan™ single nucleotide polymorphism (SNP genotyping. Methods Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates. Results TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95% were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species, however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1% error rate for TaqMan genotyping in mistakenly identifying species hybrids. Conclusion TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.

  11. Genotyping of Hepatitis C virus isolated from hepatitis patients in Southeast of Iran by taqman realtime PCR

    International Nuclear Information System (INIS)

    Farivar, T.N.; Johari, P.

    2011-01-01

    Objectives: To check TaqMan Realtime PCR in detecting genotypes of hepatitis C virus in Iran. Methods: From July 2007 to April 2009, HCV genotyping was done on 52 patients who were referred to Research Centre for infectious Disease and Tropical Medicine, in Bou-Ali Hospital, Zahedan University of Medical Sciences. All these patients had proven hepatitis C infection. Results: Out of 52 anti HCV positive samples, 28(53.84%) had genotype 1, 2 cases (3.88 %) had genotype 2 , 12 (23.08 %) had genotype 3 and 7 (13.4 %) had genotype 4 . Mixed infection with genotypes 1 and 3 was seen in 3 cases (5.77 %). Conclusion: TaqMan probes for detecting genotyping of HCV were successful in picking genotyping of HCV infection especially those with mixed genotypes. (author)

  12. A quantitative PCR (TaqMan assay for pathogenic Leptospira spp

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    Symonds Meegan L

    2002-07-01

    Full Text Available Abstract Background Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA and slide agglutination test (SAT, can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. Methods The polymerase chain reaction (PCR has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. Results and Conclusions The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

  13. Development of a Rickettsia bellii-Specific TaqMan Assay Targeting the Citrate Synthase Gene.

    Science.gov (United States)

    Hecht, Joy A; Allerdice, Michelle E J; Krawczak, Felipe S; Labruna, Marcelo B; Paddock, Christopher D; Karpathy, Sandor E

    2016-11-01

    Rickettsia bellii is a rickettsial species of unknown pathogenicity that infects argasid and ixodid ticks throughout the Americas. Many molecular assays used to detect spotted fever group (SFG) Rickettsia species do not detect R. bellii, so that infection with this bacterium may be concealed in tick populations when assays are used that screen specifically for SFG rickettsiae. We describe the development and validation of a R. bellii-specific, quantitative, real-time PCR TaqMan assay that targets a segment of the citrate synthase (gltA) gene. The specificity of this assay was validated against a panel of DNA samples that included 26 species of Rickettsia, Orientia, Ehrlichia, Anaplasma, and Bartonella, five samples of tick and human DNA, and DNA from 20 isolates of R. bellii, including 11 from North America and nine from South America. A R. bellii control plasmid was constructed, and serial dilutions of the plasmid were used to determine the limit of detection of the assay to be one copy per 4 µl of template DNA. This assay can be used to better determine the role of R. bellii in the epidemiology of tick-borne rickettsioses in the Western Hemisphere. Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.

  14. Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds.

    Science.gov (United States)

    Rojas, María; González, Isabel; Pavón, Miguel Angel; Pegels, Nicolette; Lago, Adriana; Hernández, Pablo E; García, Teresa; Martín, Rosario

    2010-06-01

    Species-specific real-time polymerase chain reaction (PCR) assays using TaqMan probes have been developed for verifying the labeling of meat and commercial meat products from game birds, including quail, pheasant, partridge, guinea fowl, pigeon, Eurasian woodcock and song thrush. The method combines the use of species-specific primers and TaqMan probes that amplify small fragments (amplicons meat products from the target species demonstrated the suitability of the assay for the detection of the target DNAs.

  15. Evaluation of a TaqMan Array Card for Detection of Central Nervous System Infections.

    Science.gov (United States)

    Onyango, Clayton O; Loparev, Vladimir; Lidechi, Shirley; Bhullar, Vinod; Schmid, D Scott; Radford, Kay; Lo, Michael K; Rota, Paul; Johnson, Barbara W; Munoz, Jorge; Oneko, Martina; Burton, Deron; Black, Carolyn M; Neatherlin, John; Montgomery, Joel M; Fields, Barry

    2017-07-01

    Infections of the central nervous system (CNS) are often acute, with significant morbidity and mortality. Routine diagnosis of such infections is limited in developing countries and requires modern equipment in advanced laboratories that may be unavailable to a number of patients in sub-Saharan Africa. We developed a TaqMan array card (TAC) that detects multiple pathogens simultaneously from cerebrospinal fluid. The 21-pathogen CNS multiple-pathogen TAC (CNS-TAC) assay includes two parasites ( Balamuthia mandrillaris and Acanthamoeba ), six bacterial pathogens ( Streptococcus pneumonia e, Haemophilus influenzae , Neisseria meningitidis , Mycoplasma pneumoniae , Mycobacterium tuberculosis , and Bartonella ), and 13 viruses (parechovirus, dengue virus, Nipah virus, varicella-zoster virus, mumps virus, measles virus, lyssavirus, herpes simplex viruses 1 and 2, Epstein-Barr virus, enterovirus, cytomegalovirus, and chikungunya virus). The card also includes human RNase P as a nucleic acid extraction control and an internal manufacturer control, GAPDH (glyceraldehyde-3-phosphate dehydrogenase). This CNS-TAC assay can test up to eight samples for all 21 agents within 2.5 h following nucleic acid extraction. The assay was validated for linearity, limit of detection, sensitivity, and specificity by using either live viruses (dengue, mumps, and measles viruses) or nucleic acid material (Nipah and chikungunya viruses). Of 120 samples tested by individual real-time PCR, 35 were positive for eight different targets, whereas the CNS-TAC assay detected 37 positive samples across nine different targets. The CNS-TAC assays showed 85.6% sensitivity and 96.7% specificity. Therefore, the CNS-TAC assay may be useful for outbreak investigation and surveillance of suspected neurological disease. Copyright © 2017 American Society for Microbiology.

  16. A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

    Science.gov (United States)

    Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

    2013-12-27

    Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

  17. TaqMan probe real-time polymerase chain reaction assay for the quantification of canine DNA in chicken nugget.

    Science.gov (United States)

    Rahman, Md Mahfujur; Hamid, Sharifah Bee Abd; Basirun, Wan Jefrey; Bhassu, Subha; Rashid, Nur Raifana Abdul; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Ali, Md Eaqub

    2016-01-01

    This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.

  18. Multiplex preamplification of specific cDNA targets prior to gene expression analysis by TaqMan Arrays

    Directory of Open Access Journals (Sweden)

    Ribal María

    2008-06-01

    Full Text Available Abstract Background An accurate gene expression quantification using TaqMan Arrays (TA could be limited by the low RNA quantity obtained from some clinical samples. The novel cDNA preamplification system, the TaqMan PreAmp Master Mix kit (TPAMMK, enables a multiplex preamplification of cDNA targets and therefore, could provide a sufficient amount of specific amplicons for their posterior analysis on TA. Findings A multiplex preamplification of 47 genes was performed in 22 samples prior to their analysis by TA, and relative gene expression levels of non-preamplified (NPA and preamplified (PA samples were compared. Overall, the mean cycle threshold (CT decrement in the PA genes was 3.85 (ranging from 2.07 to 5.01. A high correlation (r between the gene expression measurements of NPA and PA samples was found (mean r = 0.970, ranging from 0.937 to 0.994; p Conclusion We demonstrate that cDNA preamplification using the TPAMMK before TA analysis is a reliable approach to simultaneously measure gene expression of multiple targets in a single sample. Moreover, this procedure was validated in genes from degraded RNA samples and low abundance expressed genes. This combined methodology could have wide applications in clinical research, where scarce amounts of degraded RNA are usually obtained and several genes need to be quantified in each sample.

  19. Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

    Science.gov (United States)

    Pegels, N; González, I; Fernández, S; García, T; Martín, R

    2012-01-01

    A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

  20. TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

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    Ma Mingxiao

    Full Text Available Sacbrood virus (SBV is a picorna-like virus that affects honey bees (Apis mellifera and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

  1. A TaqMan Real-Time PCR Assay for Detection and Quantification of Sporisorium scitamineum in Sugarcane

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    Yachun Su

    2013-01-01

    Full Text Available Sporisorium scitamineum is a fungal smut pathogen epidemic in sugarcane producing areas. Early detection and proper identification of the smut are an essential requirement in its management practice. In this study, we developed a TaqMan real-time PCR assay using specific primers (bEQ-F/bEQ-R and a TaqMan probe (bEQ-P which were designed based on the bE (b East mating type gene (Genbank Accession no. U61290.1. This method was more sensitive (a detection limit of 10 ag pbE DNA and 0.8 ng sugarcane genomic DNA than that of conventional PCR (10 fg and 100 ng, resp.. Reliability was demonstrated through the positive detection of samples collected from artificially inoculated sugarcane plantlets (FN40. This assay was capable of detecting the smut pathogen at the initial stage (12 h of infection and suitable for inspection of sugarcane pathogen-free seed cane and seedlings. Furthermore, quantification of pathogen was verified in pathogen-challenged buds in different sugarcane genotypes, which suggested its feasibility for evaluation of smut resistance in different sugarcane genotypes. Taken together, this novel assay can be used as a diagnostic tool for sensitive, accurate, fast, and quantitative detection of the smut pathogen especially for asymptomatic seed cane or plants and evaluation of smut resistance of sugarcane genotypes.

  2. Measurement of Epstein-Barr virus DNA loads in whole blood and plasma by TaqMan PCR and in peripheral blood lymphocytes by competitive PCR.

    Science.gov (United States)

    Wadowsky, Robert M; Laus, Stella; Green, Michael; Webber, Steven A; Rowe, David

    2003-11-01

    Epstein-Barr virus (EBV) DNA load values were measured in samples of whole blood (n = 60) and plasma (n = 59) by TaqMan PCR and in samples of peripheral blood lymphocytes (PBLs) (n = 60) by competitive PCR (cPCR). The samples were obtained from 44 transplant recipients. The whole-blood and PBL loads correlated highly (r(2) > 0.900), whereas the plasma and PBL loads correlated poorly (r(2) = 0.512). Testing of whole blood by TaqMan PCR is an acceptable alternative to testing of PBLs by cPCR for quantifying EBV DNA load.

  3. Performance evaluation of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, version 2.0, for detection and quantification of hepatitis C virus RNA

    NARCIS (Netherlands)

    S.D. Pas (Suzan); R. Molenkamp (Richard); J. Schinkel (Janke); S. Rebers; C. Copra (Cederick); S. Seven-Deniz; D. Thamke (Diana); R.J. de Knegt (Robert); B.L. Haagmans (Bart); M. Schutten (Martin)

    2013-01-01

    textabstractTo evaluate the analytical performance and explore the clinical applicability of the new Roche cobas AmpliPrep/cobas TaqMan HCV test, v2.0 (CAP/CTM v2.0), a platform comparison was performed on panels and diagnostic samples with the Roche cobas AmpliPrep/cobas TaqMan HCV test (CAP/CTM

  4. CYP2D7 sequence variation interferes with TaqMan CYP2D6*15 and *35 genotyping

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    Amanda K Riffel

    2016-01-01

    Full Text Available TaqMan™ genotyping assays are widely used to genotype CYP2D6, which encodes a major drug metabolizing enzyme. Assay design for CYP2D6 can be challenging owing to the presence of two pseudogenes, CYP2D7 and CYP2D8, structural and copy number variation and numerous single nucleotide polymorphisms (SNPs some of which reflect the wild-type sequence of the CYP2D7 pseudogene. The aim of this study was to identify the mechanism causing false positive CYP2D6*15 calls and remediate those by redesigning and validating alternative TaqMan genotype assays. Among 13,866 DNA samples genotyped by the CompanionDx® lab on the OpenArray platform, 70 samples were identified as heterozygotes for 137Tins, the key SNP of CYP2D6*15. However, only 15 samples were confirmed when tested with the Luminex xTAG CYP2D6 Kit and sequencing of CYP2D6-specific long range (XL-PCR products. Genotype and gene resequencing of CYP2D6 and CYP2D7-specific XL-PCR products revealed a CC>GT dinucleotide SNP in exon 1 of CYP2D7 that reverts the sequence to CYP2D6 and allows a TaqMan assay PCR primer to bind. Because CYP2D7 also carries a Tins, a false-positive mutation signal is generated. This CYP2D7 SNP was also responsible for generating false-positive signals for rs769258 (CYP2D6*35 which is also located in exon 1. Although alternative CYP2D6*15 and *35 assays resolved the issue, we discovered a novel CYP2D6*15 subvariant in one sample that carries additional SNPs preventing detection with the alternate assay. The frequency of CYP2D6*15 was 0.1% in this ethnically diverse U.S. population sample. In addition, we also discovered linkage between the CYP2D7 CC>GT dinucleotide SNP and the 77G>A (rs28371696 SNP of CYP2D6*43. The frequency of this tentatively functional allele was 0.2%. Taken together, these findings emphasize that regardless of how careful genotyping assays are designed and evaluated before being commercially marketed, rare or unknown SNPs underneath primer and/or probe

  5. A TaqMan real-time PCR assay for detection of Meloidogyne hapla in root galls and in soil

    DEFF Research Database (Denmark)

    Sapkota, Rumakanta; Skantar, Andrea M.; Nicolaisen, Mogens

    2016-01-01

    . haplaand showed no significant amplification of DNA from non-target nematodes. The assay was able to detect M. haplain a background of plant and soil DNA. A dilution series of M. haplaeggs in soil showed a high correlation ( R 2 = 0 . 95 , P ...Early detection and quantification of Meloidogyne haplain soil is essential for effective disease management. The purpose of this study was to develop a real-time PCR assay for detection of M. haplain soil. Primers and a TaqMan probe were designed for M. hapladetection. The assay detected M......-knot development in carrots by testing soils before planting. The assay could be useful for management decisions in carrot cultivation....

  6. Development of a real-time TaqMan assay to detect mendocina sublineage Pseudomonas species in contaminated metalworking fluids.

    Science.gov (United States)

    Saha, Ratul; Donofrio, Robert S; Bagley, Susan T

    2010-08-01

    A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer-probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10(1) colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer-probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 x 10(3) and 3.9 x 10(6) CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 x 10(1) to 1.4 x 10(5) CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer-probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.

  7. Performance evaluation of the Cobas TaqMan MTB assay on respiratory specimens according to clinical application

    Directory of Open Access Journals (Sweden)

    Jong Eun Park

    2017-11-01

    Full Text Available Objective: To evaluate the performance of the Cobas TaqMan MTB assay (Cobas assay with respect to its clinical application. Methods: This was a retrospective analysis of 1154 results from 1034 patients for whom mycobacterial cultures and the Cobas assay were performed simultaneously. Based on the patient medical records, two categories of clinical application were defined: (1 the diagnosis of patients with a high probability of pulmonary tuberculosis according to clinical and radiological features (n = 128, and (2 the exclusion of tuberculosis in clinically indeterminate patients (n = 1026. Standard culture was used as the reference method. Results: The sensitivity of the Cobas assay for the detection of Mycobacterium tuberculosis was 70.4% (95% confidence interval (CI 49.7–85.5% for category 1, but only 25.0% (95% CI 4.5–64.4% for category 2. The specificity was ≥95.0% for both categories. The positive predictive value was 79.2% (95% CI 57.3–92.1% for category 1 and 33.3% (95% CI 6.0–75.9% for category 2, while the negative predictive value was 92.3% (95% CI 85.0–96.4% for category 1 and 99.4% (95% CI 98.7–99.8% for category 2. Conclusions: The results of this study indicate that Cobas assay results must be interpreted carefully according to the clinical purpose of the assay. Keywords: Mycobacterium tuberculosis, Pulmonary tuberculosis, Cobas TaqMan MTB assay, Korea

  8. Use of a Real-Time PCR TaqMan Assay for Rapid Identification and Differentiation of Burkholderia pseudomallei and Burkholderia mallei

    OpenAIRE

    U'Ren, Jana M.; Van Ert, Matthew N.; Schupp, James M.; Easterday, W. Ryan; Simonson, Tatum S.; Okinaka, Richard T.; Pearson, Talima; Keim, Paul

    2005-01-01

    A TaqMan allelic-discrimination assay designed around a synonymous single-nucleotide polymorphism was used to genotype Burkholderia pseudomallei and Burkholderia mallei isolates. The assay rapidly identifies and discriminates between these two highly pathogenic bacteria and does not cross-react with genetic near neighbors, such as Burkholderia thailandensis and Burkholderia cepacia.

  9. Comparison of allelic discrimination by dHPLC, HRM, and TaqMan in the detection of BRAF mutation V600E.

    Science.gov (United States)

    Carbonell, Pablo; Turpin, María C; Torres-Moreno, Daniel; Molina-Martínez, Irene; García-Solano, José; Perez-Guillermo, Miguel; Conesa-Zamora, Pablo

    2011-09-01

    The V600E mutation in the BRAF oncogene is associated with colorectal carcinomas, with mismatch-repair deficiency and, recently, with nonresponse to epidermal growth factor receptor inhibitor therapy. The use of reliable techniques for its detection is important. The aim of our study was to compare the performance characteristics in V600E detection of denaturing high-performance liquid chromatography (dHPLC) and high-resolution melting (HRM) with TaqMan allelic discrimination as well as direct-sequencing methods in a series of 195 colorectal paraffin-embedded specimens up to the age of 15 years. The effectiveness for obtaining results on mutation status was best using TaqMan (96.9%), followed by dHPLC (93.3%), HRM (88.7%), and sequencing (88.2%). In general, TaqMan was best for analyzing older tissues, whereas sequencing was the least efficient. Heterozygotic V600E was detected in 11.6%, 9.9%, 11.6%, and 9.9% of tissues using TaqMan, dHPLC, HRM, and sequencing, respectively. Result concordances between dHPLC and TaqMan or sequencing were excellent (κ = 0.9411 and κ = 0.8988, respectively); for HRM, the concordances were good (κ = 0.7973 and κ = 0.7488, respectively). By using DNA dilutions from tumor tissue, a minimum of 10% of V600E harboring cancer content was required for the analysis by dHPLC and HRM. dHPLC could detect four non-V600E mutations, whereas HRM detected one. Our results indicate that dHPLC and HRM are techniques that can be reliably used for the detection of the BRAFV600E mutation in archival paraffin-embedded tissues. Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  10. Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

    Science.gov (United States)

    Dinoop, K P; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R P; Narayanan, P

    2016-01-01

    Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( Pnested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

  11. Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

    Science.gov (United States)

    Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

    2013-07-01

    Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

  12. A TaqMan real-time PCR-based assay for the identification of Fasciola spp.

    Science.gov (United States)

    Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Jowers, Michael J; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

    2011-06-30

    Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR

    OpenAIRE

    Hristova, Krassimira R.; Lutenegger, Christian M.; Scow, Kate M.

    2001-01-01

    The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribos...

  14. Bat white-nose syndrome: A real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans

    Science.gov (United States)

    Laura K Muller; Jeffrey M. Lorch; Daniel L. Lindner; Michael O' Connor; Andrea Gargas; David S. Blehert

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The...

  15. Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

    Science.gov (United States)

    Kho, S L; Chua, K H; George, E; Tan, J A M A

    2013-07-15

    Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

  16. Gene Directed Enzyme Prodrug Therapy Using Rabbit Cytochrome P450 4B1 in Murine Colon Adenocarcinoma

    International Nuclear Information System (INIS)

    Kim, Sung Joo; Kang, Joo Hyun; Lee, Tae Sup; Kim, Kyeong Min; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo

    2007-01-01

    The conventional cancer therapy is chemotherapy, surgical resection and/or radiotherapy. Chemotherapy using cytotoxic drug has some problems with lack of tumor selectivity resulting in toxicity to normal tissues. To enhance the tumor selectivity of cytotoxic drug, the application of suicidal gene therapy technology was designed. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a non-toxic prodrug into a cytotoxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1- tk) and cytosine deaminase (cd). Recently, a new prodrug-converting enzyme based on rabbit cytochrome P450 4B1 gene (cyp4B1) has been reported for therapy of experimental brain tumor. This enzyme activates the prodrugs such as 4-ipomeanol (4-IM) and 2- aminoanthracene (2-AA) to highly reactive furane epoxide and unsaturated dialdehyde intermediate, respectively. DNA alkylation seems to be the main mechanism of cytotoxicity of these activated drugs. In this study, we isolated cyp4B1 cDNA from rabbit lung, transduced cyp4B1 expression vector into murine colon cancer cell, and then analyzed the cytotoxic properties of cyp4b1-activated 2-AA in cyp4B1 transduced cells to verify the cyp4B1 enzyme system for gene directed enzyme prodrug therapy

  17. Gene Directed Enzyme Prodrug Therapy Using Rabbit Cytochrome P450 4B1 in Murine Colon Adenocarcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Joo; Kang, Joo Hyun; Lee, Tae Sup; Kim, Kyeong Min; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2007-07-01

    The conventional cancer therapy is chemotherapy, surgical resection and/or radiotherapy. Chemotherapy using cytotoxic drug has some problems with lack of tumor selectivity resulting in toxicity to normal tissues. To enhance the tumor selectivity of cytotoxic drug, the application of suicidal gene therapy technology was designed. Suicidal gene therapy is based on the expression in tumor cells of a gene encoding an enzyme that converts a non-toxic prodrug into a cytotoxic product. Representative suicidal genes are Herpes simplex virus type 1 thymidine kinase (HSV1- tk) and cytosine deaminase (cd). Recently, a new prodrug-converting enzyme based on rabbit cytochrome P450 4B1 gene (cyp4B1) has been reported for therapy of experimental brain tumor. This enzyme activates the prodrugs such as 4-ipomeanol (4-IM) and 2- aminoanthracene (2-AA) to highly reactive furane epoxide and unsaturated dialdehyde intermediate, respectively. DNA alkylation seems to be the main mechanism of cytotoxicity of these activated drugs. In this study, we isolated cyp4B1 cDNA from rabbit lung, transduced cyp4B1 expression vector into murine colon cancer cell, and then analyzed the cytotoxic properties of cyp4b1-activated 2-AA in cyp4B1 transduced cells to verify the cyp4B1 enzyme system for gene directed enzyme prodrug therapy.

  18. The potential of TaqMan Array Cards for detection of multiple biological agents by real-time PCR.

    Directory of Open Access Journals (Sweden)

    Phillip A Rachwal

    Full Text Available The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels.

  19. Stratification of clear cell renal cell carcinoma (ccRCC) genomes by gene-directed copy number alteration (CNA) analysis.

    Science.gov (United States)

    Thiesen, H-J; Steinbeck, F; Maruschke, M; Koczan, D; Ziems, B; Hakenberg, O W

    2017-01-01

    Tumorigenic processes are understood to be driven by epi-/genetic and genomic alterations from single point mutations to chromosomal alterations such as insertions and deletions of nucleotides up to gains and losses of large chromosomal fragments including products of chromosomal rearrangements e.g. fusion genes and proteins. Overall comparisons of copy number alterations (CNAs) presented in 48 clear cell renal cell carcinoma (ccRCC) genomes resulted in ratios of gene losses versus gene gains between 26 ccRCC Fuhrman malignancy grades G1 (ratio 1.25) and 20 G3 (ratio 0.58). Gene losses and gains of 15762 CNA genes were mapped to 795 chromosomal cytoband loci including 280 KEGG pathways. CNAs were classified according to their contribution to Fuhrman tumour gradings G1 and G3. Gene gains and losses turned out to be highly structured processes in ccRCC genomes enabling the subclassification and stratification of ccRCC tumours in a genome-wide manner. CNAs of ccRCC seem to start with common tumour related gene losses flanked by CNAs specifying Fuhrman grade G1 losses and CNA gains favouring grade G3 tumours. The appearance of recurrent CNA signatures implies the presence of causal mechanisms most likely implicated in the pathogenesis and disease-outcome of ccRCC tumours distinguishing lower from higher malignant tumours. The diagnostic quality of initial 201 genes (108 genes supporting G1 and 93 genes G3 phenotypes) has been successfully validated on published Swiss data (GSE19949) leading to a restricted CNA gene set of 171 CNA genes of which 85 genes favour Fuhrman grade G1 and 86 genes Fuhrman grade G3. Regarding these gene sets overall survival decreased with the number of G3 related gene losses plus G3 related gene gains. CNA gene sets presented define an entry to a gene-directed and pathway-related functional understanding of ongoing copy number alterations within and between individual ccRCC tumours leading to CNA genes of prognostic and predictive value.

  20. Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay.

    Science.gov (United States)

    Chen, Jingfang; Zhang, Rusheng; Ou, Xinhua; Yao, Dong; Huang, Zheng; Li, Linzhi; Sun, Biancheng

    2017-06-01

    A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    Science.gov (United States)

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

    Science.gov (United States)

    Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

    2016-01-01

    In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

  3. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructanstructans.

    Science.gov (United States)

    Muller, Laura K.; Lorch, Jeffrey M.; Lindner, Daniel L.; O'Connor, Michael; Gargas, Andrea; Blehert, David S.

    2013-01-01

    The fungus Geomyces destructans is the causative agent of white-nose syndrome (WNS), a disease that has killed millions of North American hibernating bats. We describe a real-time TaqMan PCR test that detects DNA from G. destructans by targeting a portion of the multicopy intergenic spacer region of the rRNA gene complex. The test is highly sensitive, consistently detecting as little as 3.3 fg of genomic DNA from G. destructans. The real-time PCR test specifically amplified genomic DNA from G. destructans but did not amplify target sequence from 54 closely related fungal isolates (including 43 Geomyces spp. isolates) associated with bats. The test was further qualified by analyzing DNA extracted from 91 bat wing skin samples, and PCR results matched histopathology findings. These data indicate the real-time TaqMan PCR method described herein is a sensitive, specific, and rapid test to detect DNA from G. destructans and provides a valuable tool for WNS diagnostics and research.

  4. Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses

    DEFF Research Database (Denmark)

    Schultz, Anna Charlotte; Vega, Everado; Dalsgaard, Anders

    2011-01-01

    BackgroundCurrent detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2. ObjectiveTo develop ......Man RT-qPCR assays for the sensitive detection and direct genotyping of GI and GII NoVs from clinical and environmental matrices...... novel long-template one-step TaqMan assays (L-RT-qPCR) for the rapid detection and direct genotyping of GI and GII NoVs and to evaluate the sensitivity and specificity of the assays. Study designGI and GII-specific broadly reactive L-RT-qPCR assays were developed by combining existing NoV primers...... and probes targeting the open reading frame (ORF)1–ORF2 junction as well as region C at the 5′–ORF2. The assays were validated using GI and GII RNA transcripts and a coded panel of 75 stool samples containing NoV strains representing 9 GI genotypes and 12 GII genotypes, as well as sapoviruses, astroviruses...

  5. Development and Validation of a TaqMan Real-Time PCR Assay for the Specific Detection and Quantification of Fusarium fujikuroi in Rice Plants and Seeds.

    Science.gov (United States)

    Carneiro, Greice Amaral; Matić, Slavica; Ortu, Giuseppe; Garibaldi, Angelo; Spadaro, Davide; Gullino, Maria Lodovica

    2017-07-01

    Bakanae disease, which is caused by the seedborne pathogen Fusarium fujikuroi, is found throughout the world on rice. A TaqMan real-time PCR has been developed on the TEF 1-α gene to detect F. fujikuroi in different rice tissues. Three primer/probe sets were tested. The selected set produced an amplicon of 84 bp and was specific for F. fujikuroi with respect to eight Fusarium species of rice and six other rice common pathogens. The assay was validated for specificity, selectivity, sensitivity, repeatability, and reproducibility. The detection limit was set at 27.5 fg of DNA, which is approximately equivalent to one haploid genome of F. fujikuroi. The developed TaqMan real-time assay was able to efficiently detect and quantify F. fujikuroi from rice culms, leaves, roots, and seeds. At 1 week post-germination (wpg), the pathogen was more diffused in the green tissues, while at 3 wpg it was uniformly spread also in the roots. The highest concentration of F. fujikuroi was measured in the M6 cultivar, which showed around 1,450 fungal cells/g. The assay was sufficiently sensitive to detect a few genomic equivalents in the rice seeds, corresponding to 9.89 F. fujikuroi cells/g. The assay permitted bakanae disease to be detected in asymptomatic tissues at the early rice development stages.

  6. Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

    Science.gov (United States)

    Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

    2011-01-01

    Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus. PMID:22363089

  7. Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR "Double Check" Strategy

    DEFF Research Database (Denmark)

    Hoffmann, B.; Freuling, C. M.; Wakeley, P. R.

    2010-01-01

    To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus...... sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany......), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome...

  8. Designing a time-effective TaqMan probe-based real-time polymerase chain reaction protocol for the identification of Yersinia enterocolitica in raw pork meat

    Directory of Open Access Journals (Sweden)

    Milena Alicja Stachelska

    2017-01-01

    Full Text Available The aim of this study was to design a time-effective method comprising a short pre-enrichment step in a non-selective broth in combination with the TaqMan probe applied in the real-time polymerase chain reaction to detect Yersinia enterocolitica strains in raw pork meat. The method enabled to detect 1 colony forming unit per 25 mg of Yersinia enterocolitica in pork meat. The specificity and reliability of the method was not diminished by the company of microflora naturally present in meat. The method was found successful to detect pathogenic Yersinia enterocolitica strains in pork meat. It is advised to be used for assessing the microbial risk and for controlling the microbial quality of meat and meat products.

  9. Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus.

    Science.gov (United States)

    Liu, Jie; Ochieng, Caroline; Wiersma, Steve; Ströher, Ute; Towner, Jonathan S; Whitmer, Shannon; Nichol, Stuart T; Moore, Christopher C; Kersh, Gilbert J; Kato, Cecilia; Sexton, Christopher; Petersen, Jeannine; Massung, Robert; Hercik, Christine; Crump, John A; Kibiki, Gibson; Maro, Athanasia; Mujaga, Buliga; Gratz, Jean; Jacob, Shevin T; Banura, Patrick; Scheld, W Michael; Juma, Bonventure; Onyango, Clayton O; Montgomery, Joel M; Houpt, Eric; Fields, Barry

    2016-01-01

    Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2.5 h from sample to results and can simultaneously detect 26 AFI-associated organisms, including 15 viruses (chikungunya, Crimean-Congo hemorrhagic fever [CCHF] virus, dengue, Ebola virus, Bundibugyo virus, Sudan virus, hantaviruses [Hantaan and Seoul], hepatitis E, Marburg, Nipah virus, o'nyong-nyong virus, Rift Valley fever virus, West Nile virus, and yellow fever virus), 8 bacteria (Bartonella spp., Brucella spp., Coxiella burnetii, Leptospira spp., Rickettsia spp., Salmonella enterica and Salmonella enterica serovar Typhi, and Yersinia pestis), and 3 protozoa (Leishmania spp., Plasmodium spp., and Trypanosoma brucei). Two extrinsic controls (phocine herpesvirus 1 and bacteriophage MS2) were included to ensure extraction and amplification efficiency. Analytical validation was performed on spiked specimens for linearity, intra-assay precision, interassay precision, limit of detection, and specificity. The performance of the card on clinical specimens was evaluated with 1,050 blood samples by comparison to the individual real-time PCR assays, and the TAC exhibited an overall 88% (278/315; 95% confidence interval [CI], 84% to 92%) sensitivity and a 99% (5,261/5,326, 98% to 99%) specificity. This TaqMan array card can be used in field settings as a rapid screen for outbreak investigation or for the surveillance of pathogens, including Ebola virus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Development of a TaqMan Allelic Discrimination Assay for detection of Single Nucleotides Polymorphisms associated with anti-malarial drug resistance

    Directory of Open Access Journals (Sweden)

    Kamau Edwin

    2012-01-01

    Full Text Available Abstract Background Anti-malarial drug resistance poses a threat to current global efforts towards control and elimination of malaria. Several methods are used in monitoring anti-malarial drug resistance. Molecular markers such as single nucleotide polymorphism (SNP for example are increasingly being used to identify genetic mutations related to anti-malarial drug resistance. Several methods are currently being used in analysis of SNP associated with anti-malarial drug resistance and although each one of these methods has unique strengths and shortcoming, there is still need to improve and/or develop new methods that will close the gap found in the current methods. Methods TaqMan Allelic Discrimination assays for detection of SNPs associated with anti-malarial drug resistance were designed for analysis on Applied Biosystems PCR platform. These assays were designed by submitting SNP sequences associated with anti-malarial drug resistance to Applied Biosystems website. Eleven SNPs associated with resistance to anti-malarial drugs were selected and tested. The performance of each SNP assay was tested by creating plasmid DNAs carrying codons of interests and analysing them for analysis. To test the sensitivity and specificity of each SNP assay, 12 clinical samples were sequenced at codons of interest and used in the analysis. Plasmid DNAs were used to establish the Limit of Detection (LoD for each assay. Results Data from genetic profiles of the Plasmodium falciparum laboratory strains and sequence data from 12 clinical samples was used as the reference method with which the performance of the SNP assays were compared to. The sensitivity and specificity of each SNP assay was establish at 100%. LoD for each assay was established at 2 GE, equivalent to less than 1 parasite/μL. SNP assays performed well in detecting mixed infection and analysis of clinical samples. Conclusion TaqMan Allelic Discrimination assay provides a good alternative tool in

  11. Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

    Science.gov (United States)

    Wang, Li-Ping; Lei, Kun

    2016-12-01

    Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

  12. TaqMan real-time PCR assays for single-nucleotide polymorphisms which identify Francisella tularensis and its subspecies and subpopulations.

    Directory of Open Access Journals (Sweden)

    Dawn N Birdsell

    Full Text Available Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis, therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays

  13. Results of the Abbott RealTime HIV-1 Assay for Specimens Yielding “Target Not Detected” Results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test▿

    OpenAIRE

    Babady, N. Esther; Germer, Jeffrey J.; Yao, Joseph D. C.

    2009-01-01

    No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding “target not detected” results by CTM.

  14. Results of the Abbott RealTime HIV-1 assay for specimens yielding "target not detected" results by the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test.

    Science.gov (United States)

    Babady, N Esther; Germer, Jeffrey J; Yao, Joseph D C

    2010-03-01

    No significantly discordant results were observed between the Abbott RealTime HIV-1 assay and the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test (CTM) among 1,190 unique clinical plasma specimens obtained from laboratories located in 40 states representing all nine U.S. geographic regions and previously yielding "target not detected" results by CTM.

  15. Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Takao Ito

    Full Text Available Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

  16. Quantification of the biocontrol agent Trichoderma harzianum with real-time TaqMan PCR and its potential extrapolation to the hyphal biomass.

    Science.gov (United States)

    López-Mondéjar, Rubén; Antón, Anabel; Raidl, Stefan; Ros, Margarita; Pascual, José Antonio

    2010-04-01

    The species of the genus Trichoderma are used successfully as biocontrol agents against a wide range of phytopathogenic fungi. Among them, Trichoderma harzianum is especially effective. However, to develop more effective fungal biocontrol strategies in organic substrates and soil, tools for monitoring the control agents are required. Real-time PCR is potentially an effective tool for the quantification of fungi in environmental samples. The aim of this study consisted of the development and application of a real-time PCR-based method to the quantification of T. harzianum, and the extrapolation of these data to fungal biomass values. A set of primers and a TaqMan probe for the ITS region of the fungal genome were designed and tested, and amplification was correlated to biomass measurements obtained with optical microscopy and image analysis, of the hyphal length of the mycelium of the colony. A correlation of 0.76 between ITS copies and biomass was obtained. The extrapolation of the quantity of ITS copies, calculated based on real-time PCR data, into quantities of fungal biomass provides potentially a more accurate value of the quantity of soil fungi. Copyright 2009 Elsevier Ltd. All rights reserved.

  17. Characterization of Phytophthora nicotianae isolates in southeast Spain and their detection and quantification through a real-time TaqMan PCR.

    Science.gov (United States)

    Blaya, Josefa; Lacasa, Carmen; Lacasa, Alfredo; Martínez, Victoriano; Santísima-Trinidad, Ana B; Pascual, Jose A; Ros, Margarita

    2015-04-01

    The soil-borne pathogens Phytophthora nicotianae and P. capsici are the causal agents of root and stem rot of many plant species. Although P. capsici was considered the causal agent in one of the main pepper production areas of Spain to date, evidence of the presence of P. nicotianae was found. We aimed to survey the presence of P. nicotianae and study the variability in its populations in this area in order to improve the management of Tristeza disease. A new specific primer and a TaqMan probe were designed based on the internal transcribed spacer regions of ribosomal DNA to detect and quantify P. nicotianae. Both morphological and molecular analysis showed its presence and confirmed it to be the causal agent of the Phytophthora disease symptoms in the studied area. The genetic characterization among P. nicotianae populations showed a low variability of genetic diversity among the isolates. Only isolates of the A2 mating type were detected. Not only is a specific and early detection of P. nicotianae essential but also the study of genetic variability among isolates for the appropriate management of the disease, above all, in producing areas with favorable conditions for the advance of the disease. © 2014 Society of Chemical Industry.

  18. Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams

    Directory of Open Access Journals (Sweden)

    Adeline Bidault

    2015-12-01

    Full Text Available The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD in the Manila clam Venerupis (=Ruditapes philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600T V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW or extrapallial fluids (EF samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 101 bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.

  19. Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test.

    Science.gov (United States)

    Kim, Hanah; Hur, Mina; Bae, Eunsin; Lee, Kyung-A; Lee, Woo-In

    2018-02-19

    Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0-1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

  20. Association Between Stool Enteropathogen Quantity and Disease in Tanzanian Children Using TaqMan Array Cards: A Nested Case-Control Study

    Science.gov (United States)

    Platts-Mills, James A.; Gratz, Jean; Mduma, Esto; Svensen, Erling; Amour, Caroline; Liu, Jie; Maro, Athanasia; Saidi, Queen; Swai, Ndealilia; Kumburu, Happiness; McCormick, Benjamin J. J.; Kibiki, Gibson; Houpt, Eric R.

    2014-01-01

    Etiologic studies of diarrhea are limited by uneven diagnostic methods and frequent asymptomatic detection of enteropathogens. Polymerase chain reaction-based stool pathogen quantification may help distinguish clinically significant infections. We performed a nested case-control study of diarrhea in infants from a community-based birth cohort in Tanzania. We tested 71 diarrheal samples and pre-diarrheal matched controls with a laboratory-developed TaqMan Array Card for 19 enteropathogens. With qualitative detection, no pathogens were significantly associated with diarrhea. When pathogen quantity was considered, rotavirus (odds ratio [OR] = 2.70 per log10 increase, P < 0.001), astrovirus (OR = 1.49, P = 0.01), and Shigella/enteroinvasive Escherichia coli (OR = 1.47, P = 0.04) were associated with diarrhea. Enterotoxigenic E. coli (0.15 SD decline in length-for-age z score after 3 months per log10 increase, P < 0.001) and Campylobacter jejuni/C. coli (0.11 SD decline, P = 0.003) in pre-diarrheal stools were associated with poor linear growth. Quantitative analysis can help refine the association between enteropathogens and disease in endemic settings. PMID:24189366

  1. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  2. Quantitative Tetraplex Real-Time Polymerase Chain Reaction Assay with TaqMan Probes Discriminates Cattle, Buffalo, and Porcine Materials in Food Chain.

    Science.gov (United States)

    Hossain, M A Motalib; Ali, Md Eaqub; Sultana, Sharmin; Asing; Bonny, Sharmin Quazi; Kader, Md Abdul; Rahman, M Aminur

    2017-05-17

    Cattle, buffalo, and porcine materials are widely adulterated, and their quantification might safeguard health, religious, economic, and social sanctity. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays have been documented but they are just suitable for identification, cannot quantify adulterations. We described here a quantitative tetraplex real-time PCR assay with TaqMan Probes to quantify contributions from cattle, buffalo, and porcine materials simultaneously. Amplicon-sizes were very short (106-, 90-, and 146-bp for cattle, buffalo, and porcine) because longer targets could be broken down, bringing serious ambiguity in molecular diagnostics. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 27 frankfurters and 27 meatballs reflected 84-115% target recovery at 0.1-10% adulterations. Finally, a test of 36 commercial products revealed 71% beef frankfurters, 100% meatballs, and 85% burgers contained buffalo adulteration, but no porcine was found in beef products.

  3. Rapid identification of tomato Sw-5 resistance-breaking isolates of Tomato spotted wilt virus using high resolution melting and TaqMan SNP Genotyping assays as allelic discrimination techniques.

    Directory of Open Access Journals (Sweden)

    Valentina di Rienzo

    Full Text Available In tomato, resistance to Tomato spotted wilt virus (TSWV is conferred by the dominant gene, designated Sw-5. Virulent Sw-5 resistance breaking (SRB mutants of TSWV have been reported on Sw-5 tomato cultivars. Two different PCR-based allelic discrimination techniques, namely Custom TaqMan™ SNP Genotyping and high-resolution melting (HRM assays, were developed and compared for their ability to distinguish between avirulent (Sw-5 non-infecting, SNI and SRB biotypes. TaqMan assays proved to be more sensitive (threshold of detection in a range of 50-70 TSWV RNA copies and more reliable than HRM, assigning 25 TSWV isolates to their correct genotype with an accuracy of 100%. Moreover, the TaqMan SNP assays were further improved developing a rapid and simple protocol that included crude leaf extraction for RNA template preparations. On the other hand, HRM assays showed higher levels of sensitivity than TaqMan when used to co-detect both biotypes in different artificial mixtures. These diagnostic assays contributed to gain preliminary information on the epidemiology of TSWV isolates in open field conditions. In fact, the presented data suggest that SRB isolates are present as stable populations established year round, persisting on both winter (globe artichoke and summer (tomato crops, in the same cultivated areas of Southern Italy.

  4. Mouse Mammary Tumor Virus Promoter-Containing Retroviral Promoter Conversion Vectors for Gene-Directed Enzyme Prodrug Therapy are Functional in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Reinhard Klein

    2008-01-01

    Full Text Available Gene directed-enzyme prodrug therapy (GDEPT is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1 metabolizes the prodrugs cyclophosphamide (CPA and ifosfamide (IFA to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC50 values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

  5. The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Green, Laura K.; Storey, Mathew A. [School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140 (New Zealand); Williams, Elsie M. [School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140 (New Zealand); Victoria University Centre for Biodiscovery, School of Biological Sciences, Victoria University of Wellington, Wellington 6140 (New Zealand); Patterson, Adam V.; Smaill, Jeff B. [Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1142 (New Zealand); Auckland Cancer Society Research Centre, University of Auckland, Grafton, Auckland 1142 (New Zealand); Copp, Janine N.; Ackerley, David F., E-mail: david.ackerley@vuw.ac.nz [School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington 6140 (New Zealand); Victoria University Centre for Biodiscovery, School of Biological Sciences, Victoria University of Wellington, Wellington 6140 (New Zealand); Maurice Wilkins Centre for Molecular Biodiscovery, School of Biological Sciences, University of Auckland, Auckland 1142 (New Zealand)

    2013-08-08

    Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the “gold standard” nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT.

  6. The Flavin Reductase MsuE Is a Novel Nitroreductase that Can Efficiently Activate Two Promising Next-Generation Prodrugs for Gene-Directed Enzyme Prodrug Therapy

    International Nuclear Information System (INIS)

    Green, Laura K.; Storey, Mathew A.; Williams, Elsie M.; Patterson, Adam V.; Smaill, Jeff B.; Copp, Janine N.; Ackerley, David F.

    2013-01-01

    Bacterial nitroreductase enzymes that can efficiently catalyse the oxygen-independent reduction of prodrugs originally developed to target tumour hypoxia offer great potential for expanding the therapeutic range of these molecules to aerobic tumour regions, via the emerging cancer strategy of gene-directed enzyme prodrug therapy (GDEPT). Two promising hypoxia prodrugs for GDEPT are the dinitrobenzamide mustard PR-104A, and the nitrochloromethylbenzindoline prodrug nitro-CBI-DEI. We describe here use of a nitro-quenched fluorogenic probe to identify MsuE from Pseudomonas aeruginosa as a novel nitroreductase candidate for GDEPT. In SOS and bacteria-delivered enzyme prodrug cytotoxicity assays MsuE was less effective at activating CB1954 (a first-generation GDEPT prodrug) than the “gold standard” nitroreductases NfsA and NfsB from Escherichia coli. However, MsuE exhibited comparable levels of activity with PR-104A and nitro-CBI-DEI, and is the first nitroreductase outside of the NfsA and NfsB enzyme families to do so. These in vitro findings suggest that MsuE is worthy of further evaluation in in vivo models of GDEPT

  7. Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

    Science.gov (United States)

    Werz, Christian; Lee, Tom V.; Lee, Peter L.; Lackey, Melinda; Bolduc, Clare; Stein, David S.; Bergmann, Andreas

    2009-01-01

    Summary Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation. PMID:16280349

  8. Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide

    Directory of Open Access Journals (Sweden)

    Yuexia Wang

    2015-09-01

    Full Text Available Real-time polymerase chain reaction (PCR allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at −18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 103 CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 100 CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach.

  9. TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease.

    Science.gov (United States)

    Bohuski, Elizabeth; Lorch, Jeffrey M; Griffin, Kathryn M; Blehert, David S

    2015-04-15

    Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease. We developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targets the internal transcribed spacer region (ITS) of the fungal genome while the other targets the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required. These assays represent the first published methods to detect O. ophiodiicola by real-time PCR. The ITS assay has great utility for assisting with SFD diagnoses whereas the IGS assay offers a valuable tool for research-based applications.

  10. Detection of viable Salmonella in ice cream by TaqMan real-time polymerase chain reaction assay combining propidium monoazide.

    Science.gov (United States)

    Wang, Yuexia; Yang, Ming; Liu, Shuchun; Chen, Wanyi; Suo, Biao

    2015-09-01

    Real-time polymerase chain reaction (PCR) allows rapid detection of Salmonella in frozen dairy products, but it might cause a false positive detection result because it might amplify DNA from dead target cells as well. In this study, Salmonella-free frozen ice cream was initially inoculated with heat-killed Salmonella Typhimurium cells and stored at -18°C. Bacterial DNA extracted from the sample was amplified using TaqMan probe-based real-time PCR targeting the invA gene. Our results indicated that DNA from the dead cells remained stable in frozen ice cream for at least 20 days, and could produce fluorescence signal for real-time PCR as well. To overcome this limitation, propidium monoazide (PMA) was combined with real-time PCR. PMA treatment can effectively prevent PCR amplification from heat-killed Salmonella cells in frozen ice cream. The PMA real-time PCR assay can selectively detect viable Salmonella at as low as 10 3  CFU/mL. Combining 18 hours of pre-enrichment with the assay allows for the detection of viable Salmonella at 10 0  CFU/mL and avoiding the false-positive result of dead cells. The PMA real-time PCR assay provides an alternative specifically for detection of viable Salmonella in ice cream. However, when the PMA real-time PCR assay was evaluated in ice cream subjected to frozen storage, it obviously underestimated the contamination situation of viable Salmonella, which might lead to a false negative result. According to this result, the use of enrichment prior to PMA real-time PCR analysis remains as the more appropriate approach. Copyright © 2015. Published by Elsevier B.V.

  11. Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens.

    Science.gov (United States)

    Qu, X S; Wanner, L A; Christ, B J

    2011-03-01

    To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. Real-time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C(t) values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. This multiplex real-time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  12. Search Engine for Antimicrobial Resistance: A Cloud Compatible Pipeline and Web Interface for Rapidly Detecting Antimicrobial Resistance Genes Directly from Sequence Data.

    Science.gov (United States)

    Rowe, Will; Baker, Kate S; Verner-Jeffreys, David; Baker-Austin, Craig; Ryan, Jim J; Maskell, Duncan; Pearce, Gareth

    2015-01-01

    Antimicrobial resistance remains a growing and significant concern in human and veterinary medicine. Current laboratory methods for the detection and surveillance of antimicrobial resistant bacteria are limited in their effectiveness and scope. With the rapidly developing field of whole genome sequencing beginning to be utilised in clinical practice, the ability to interrogate sequencing data quickly and easily for the presence of antimicrobial resistance genes will become increasingly important and useful for informing clinical decisions. Additionally, use of such tools will provide insight into the dynamics of antimicrobial resistance genes in metagenomic samples such as those used in environmental monitoring. Here we present the Search Engine for Antimicrobial Resistance (SEAR), a pipeline and web interface for detection of horizontally acquired antimicrobial resistance genes in raw sequencing data. The pipeline provides gene information, abundance estimation and the reconstructed sequence of antimicrobial resistance genes; it also provides web links to additional information on each gene. The pipeline utilises clustering and read mapping to annotate full-length genes relative to a user-defined database. It also uses local alignment of annotated genes to a range of online databases to provide additional information. We demonstrate SEAR's application in the detection and abundance estimation of antimicrobial resistance genes in two novel environmental metagenomes, 32 human faecal microbiome datasets and 126 clinical isolates of Shigella sonnei. We have developed a pipeline that contributes to the improved capacity for antimicrobial resistance detection afforded by next generation sequencing technologies, allowing for rapid detection of antimicrobial resistance genes directly from sequencing data. SEAR uses raw sequencing data via an intuitive interface so can be run rapidly without requiring advanced bioinformatic skills or resources. Finally, we show that SEAR

  13. Controlling feeding behavior by chemical or gene-directed targeting in the brain: What’s so spatial about our methods?

    Directory of Open Access Journals (Sweden)

    Arshad M Khan

    2013-12-01

    Full Text Available Intracranial chemical injection (ICI methods have been used to identify the locations in the brain where feeding behavior can be controlled acutely. Scientists conducting ICI studies often document their injection site locations, thereby leaving kernels of valuable location data for others to use to further characterize feeding control circuits. Unfortunately, this rich dataset has not yet been formally contextualized with other published neuroanatomical data. In particular, axonal tracing studies have delineated several neural circuits originating in the same areas where ICI injection feeding-control sites have been documented, but it remains unclear whether these circuits participate in feeding control. However, comparing injection sites with other types of location data requires careful anatomical registration between the datasets. Here, a conceptual framework is presented for how such anatomical registration efforts can be performed. For example, by using a simple atlas alignment tool, a hypothalamic locus sensitive to the orexigenic effects of neuropeptide Y (NPY can be aligned accurately with the locations of neurons labeled by anterograde tracers or those known to express NPY receptors or feeding-related peptides. This approach can also be applied to those intracranial gene-directed injection (IGI methods (e.g., site-specific recombinase methods, RNA expression or interference, optogenetics and pharmacosynthetics that involve viral injections to targeted neuronal populations. Spatial alignment efforts can be accelerated if location data from ICI/IGI methods are mapped to stereotaxic brain atlases to allow powerful neuroinformatics tools to overlay different types of data in the same reference space. Atlas-based mapping will be critical for community-based sharing of location data for feeding control circuits, and will accelerate our understanding of structure-function relationships in the brain for mammalian models of obesity and

  14. Cross-platform comparison of SYBR® Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC study

    Directory of Open Access Journals (Sweden)

    Dial Stacey L

    2008-07-01

    Full Text Available Abstract Background The MicroArray Quality Control (MAQC project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006. The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta RT-PCR™ and QuantiGene®. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR® Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profiler™ PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. Results The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. Conclusion These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

  15. Development of TaqMan probes targeting the four major celiac disease epitopes found in α-gliadin sequences of spelt (Triticum aestivum ssp. spelta) and bread wheat (Triticum aestivum ssp. aestivum).

    Science.gov (United States)

    Dubois, Benjamin; Bertin, Pierre; Muhovski, Yordan; Escarnot, Emmanuelle; Mingeot, Dominique

    2017-01-01

    Celiac disease (CD) is caused by specific sequences of gluten proteins found in cereals such as bread wheat ( Triticum aestivum ssp. aestivum ) and spelt ( T. aestivum ssp. spelta ). Among them, the α-gliadins display the highest immunogenicity, with four T-cell stimulatory epitopes. The toxicity of each epitope sequence can be reduced or even suppressed according to the allelic form of each sequence. One way to address the CD problem would be to make use of this allelic variability in breeding programs to develop safe varieties, but tools to track the presence of toxic epitopes are required. The objective of this study was to develop a tool to accurately detect and quantify the immunogenic content of expressed α-gliadins of spelt and bread wheat. Four TaqMan probes that only hybridize to the canonical-i.e. toxic-form of each of the four epitopes were developed and their specificity was demonstrated. Six TaqMan probes targeting stable reference genes were also developed and constitute a tool to normalize qPCR data. The probes were used to measure the epitope expression levels of 11 contrasted spelt accessions and three ancestral diploid accessions of bread wheat and spelt. A high expression variability was highlighted among epitopes and among accessions, especially in Asian spelts, which showed lower epitope expression levels than the other spelts. Some discrepancies were identified between the canonical epitope expression level and the global amount of expressed α-gliadins, which makes the designed TaqMan probes a useful tool to quantify the immunogenic potential independently of the global amount of expressed α-gliadins. The results obtained in this study provide useful tools to study the immunogenic potential of expressed α-gliadin sequences from Triticeae accessions such as spelt and bread wheat. The application of the designed probes to contrasted spelt accessions revealed a high variability and interesting low canonical epitope expression levels in the

  16. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

    Science.gov (United States)

    Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

    2015-01-01

    Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

  17. Comparison of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 test v1.0 with v2.0 in HIV-1 viral load quantification

    Directory of Open Access Journals (Sweden)

    Yi-Ching Tung

    2015-04-01

    Full Text Available Roche modified the COBAS AmpliPrep/COBAS TaqMan human immunodeficiency virus type 1 (HIV-1 test version 1.0 (CAP/CTM v1.0, resulting in the COBAS AmpliPrep/COBAS TaqMan HIV-1 test version 2.0 (CAP/CTM v2.0. The aim of this study was to evaluate the performance of the CAP/CTM v2.0 and to compare this performance with that of the CAP/CTM v1.0. The study was conducted in a small local study group in Kaohsiung Medical University Hospital, Kaohsiung, Taiwan. A total of 86 plasma samples from HIV-1-seropositive patients were tested using the two assays. The correlation and concordance of results between the two assays were calculated. The CAP/CTM v2.0 generated higher values than did the CAP/CTM v1.0, and five samples (5.8% yielded a difference of > 1 log10 copies/mL. In addition, our data show that CAP/CTM v1.0 and CAP/CTM v2.0 yielded relatively consistent values for 23 samples with low viral loads (< 200 copies/mL. Furthermore, when viral loads were in a medium range (2–5 log10 copies/mL, the results of the two assays were more compatible. This study shows a good correlation between CAP/CTM v1.0 and v2.0 in HIV-1 viral load measurement. Further attention must be paid to those cases in which measured viral loads present larger differences between the two assays.

  18. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

    Energy Technology Data Exchange (ETDEWEB)

    Roeder, Martin; Vieths, Stefan [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany); Holzhauser, Thomas, E-mail: holth@pei.de [Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen (Germany)

    2011-01-24

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg{sup -1} almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg{sup -1}. We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg{sup -1} almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg{sup -1}. Further, between 100 and 100,000 mg kg{sup -1} spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a

  19. TaqMan探针荧光定量PCR检测花生油中掺入棕榈油的研究%Determination of palm oil adulterated in peanut oil with the TaqMan probe -based RT- PCR method

    Institute of Scientific and Technical Information of China (English)

    周慧; 梁宇斌; 吴苏喜; 李晓明; 裴伟; 杨涛

    2011-01-01

    The method of Real - time fluorescence quantitative polymerase chain reaction ( RT - PCR) with TaqMan fluorescent probe was chosen to fast detect the amount of palm oil mixed in peanut oil. MT3 - B gene of palm was selected as target gene to detect palm oil from peanut oil. The primers of MT3 - B and TaqMan probe were designed, MT3 - B gene reconstructed plasmid was built as absolute quantitative criteria for quantitative RT - PCR to establish standard curve. Peanut oil blended with 1% -40% concentration gradient palm oil was extracted DNA to test palm content by RT - PCR. The result showed that the correlation coefficient (R1) of standard curve with logarithmic linear regression analysis was 0.996. When the adulteration of palm oil in peanut oil reached 5% volume, MT3 - B gene of 17.431 copies per milli-liter of mixed oil could be detected . The method showed good sensitivity, specificity and repeatability.%根据棕榈内源基因MT3 -B设计引物和TaqMan探针,采用基因重组技术构建用于检测棕榈基因MT3 -B的重组质粒作为绝对定量标准品,建立标准曲线,对花生油中掺入棕榈油1% ~40%梯度混合油品提取DNA进行棕榈成分定量检测.结果表明,重组质粒标准品荧光定量标准曲线对数线性回归分析相关系数(R2)为0.996;花生油中掺入棕榈油达到5%时,可检出每亳升混合油品中棕榈MT3 -B基因17.431 copies,检测的重复性和特异性好.

  20. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay.

    Science.gov (United States)

    Röder, Martin; Vieths, Stefan; Holzhauser, Thomas

    2011-01-24

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(®) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(®) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

  1. Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Roeder, Martin; Vieths, Stefan; Holzhauser, Thomas

    2011-01-01

    Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg -1 almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg -1 . We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg -1 almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg -1 . Further, between 100 and 100,000 mg kg -1 spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and

  2. Evaluation of the Aptima HBV Quant assay vs. the COBAS TaqMan HBV test using the high pure system for the quantitation of HBV DNA in plasma and serum samples.

    Science.gov (United States)

    Schalasta, Gunnar; Börner, Anna; Speicher, Andrea; Enders, Martin

    2018-03-28

    Proper management of patients with chronic hepatitis B virus (HBV) infection requires monitoring of plasma or serum HBV DNA levels using a highly sensitive nucleic acid amplification test. Because commercially available assays differ in performance, we compared herein the performance of the Hologic Aptima HBV Quant assay (Aptima) to that of the Roche Cobas TaqMan HBV test for use with the high pure system (HPS/CTM). Assay performance was assessed using HBV reference panels as well as plasma and serum samples from chronically HBV-infected patients. Method correlation, analytical sensitivity, precision/reproducibility, linearity, bias and influence of genotype were evaluated. Data analysis was performed using linear regression, Deming correlation analysis and Bland-Altman analysis. Agreement between the assays for the two reference panels was good, with a difference in assay values vs. target 0.98). The two assays had similar bias and precision across the different genotypes tested at low viral loads (25-1000 IU/mL). Aptima has a performance comparable with that of HPS/CTM, making it suitable for use for HBV infection monitoring. Aptima runs on a fully automated platform (the Panther system) and therefore offers a significantly improved workflow compared with HPS/CTM.

  3. New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

    Science.gov (United States)

    Fernandes-Monteiro, Alice G; Trindade, Gisela F; Yamamura, Anna M Y; Moreira, Otacilio C; de Paula, Vanessa S; Duarte, Ana Cláudia M; Britto, Constança; Lima, Sheila Maria B

    2015-01-01

    The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

  4. Improving clinical laboratory efficiency: a time-motion evaluation of the Abbott m2000 RealTime and Roche COBAS AmpliPrep/COBAS TaqMan PCR systems for the simultaneous quantitation of HIV-1 RNA and HCV RNA.

    Science.gov (United States)

    Amendola, Alessandra; Coen, Sabrina; Belladonna, Stefano; Pulvirenti, F Renato; Clemens, John M; Capobianchi, M Rosaria

    2011-08-01

    Diagnostic laboratories need automation that facilitates efficient processing and workflow management to meet today's challenges for expanding services and reducing cost, yet maintaining the highest levels of quality. Processing efficiency of two commercially available automated systems for quantifying HIV-1 and HCV RNA, Abbott m2000 system and Roche COBAS Ampliprep/COBAS TaqMan 96 (docked) systems (CAP/CTM), was evaluated in a mid/high throughput workflow laboratory using a representative daily workload of 24 HCV and 72 HIV samples. Three test scenarios were evaluated: A) one run with four batches on the CAP/CTM system, B) two runs on the Abbott m2000 and C) one run using the Abbott m2000 maxCycle feature (maxCycle) for co-processing these assays. Cycle times for processing, throughput and hands-on time were evaluated. Overall processing cycle time was 10.3, 9.1 and 7.6 h for Scenarios A), B) and C), respectively. Total hands-on time for each scenario was, in order, 100.0 (A), 90.3 (B) and 61.4 min (C). The interface of an automated analyzer to the laboratory workflow, notably system set up for samples and reagents and clean up functions, are as important as the automation capability of the analyzer for the overall impact to processing efficiency and operator hands-on time.

  5. Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

    Directory of Open Access Journals (Sweden)

    Puisieux Alain

    2003-10-01

    Full Text Available Abstract Background Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive. Results We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC in peripheral blood mononuclear cells; the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy. Conclusion Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.

  6. Smallpox and pan-orthopox virus detection by real-time 3'-minor groove binder TaqMan assays on the roche LightCycler and the Cepheid smart Cycler platforms.

    Science.gov (United States)

    Kulesh, David A; Baker, Robert O; Loveless, Bonnie M; Norwood, David; Zwiers, Susan H; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P; Huggins, John; Jahrling, Peter B

    2004-02-01

    We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3'-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 x 10(7), 1.24 x 10(5), 1.24 x 10(3), and 1.24 x 10(1) genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs

  7. Smallpox and pan-Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

    Science.gov (United States)

    Kulesh, David A.; Baker, Robert O.; Loveless, Bonnie M.; Norwood, David; Zwiers, Susan H.; Mucker, Eric; Hartmann, Chris; Herrera, Rafael; Miller, David; Christensen, Deanna; Wasieloski, Leonard P.; Huggins, John; Jahrling, Peter B.

    2004-01-01

    We designed, optimized, and extensively tested several sensitive and specific real-time PCR assays for rapid detection of both smallpox and pan-orthopox virus DNAs. The assays are based on TaqMan 3′-minor groove binder chemistry and were performed on both the rapid-cycling Roche LightCycler and the Cepheid Smart Cycler platforms. The hemagglutinin (HA) J7R, B9R, and B10R genes were used as targets for the variola virus-specific assays, and the HA and DNA polymerase-E9L genes were used as targets for the pan-orthopox virus assays. The five orthopox virus assays were tested against a panel of orthopox virus DNAs (both genomic and cloned) at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). The results indicated that each assay was capable of detecting both the appropriate cloned gene and genomic DNA. The assays showed no cross-reactivity to the 78 DNAs in the USAMRIID bacterial cross-reactivity panel. The limit of detection (LOD) of each assay was determined to be between 12 and 25 copies of target DNA. The assays were also run against a blind panel of DNAs at the Centers for Disease Control and Prevention (CDC) on both the LightCycler and the Smart Cycler. The panel consisted of eight different variola virus isolates, five non-variola virus orthopox virus isolates, two varicella-zoster virus isolates, and one herpes simplex virus isolate. Each sample was tested in triplicate at 2.5 ng, 25 pg, 250 fg, and 2.5 fg, which represent 1.24 × 107, 1.24 × 105, 1.24 × 103, and 1.24 × 101 genome equivalents, respectively. The results indicated that each of the five assays was 100% specific (no false positives) when tested against both the USAMRIID panels and the CDC blind panel. With the CDC blind panel, the LightCycler was capable of detecting 96.2% of the orthopox virus DNAs and 93.8% of the variola virus DNAs. The Smart Cycler was capable of detecting 92.3% of the orthopox virus DNAs and between 75 and 93.8% of the variola virus DNAs. However

  8. Evaluation of the performance of Abbott m2000 and Roche COBAS Ampliprep/COBAS Taqman assays for HIV-1 viral load determination using dried blood spots and dried plasma spots in Kenya.

    Science.gov (United States)

    Zeh, Clement; Ndiege, Kenneth; Inzaule, Seth; Achieng, Rebecca; Williamson, John; Chih-Wei Chang, Joy; Ellenberger, Dennis; Nkengasong, John

    2017-01-01

    Routine HIV viral load testing is not widely accessible in most resource-limited settings, including Kenya. To increase access to viral load testing, alternative sample types like dried blood spots (DBS), which overcome the logistic barriers associated with plasma separation and cold chain shipment need to be considered and evaluated. The current study evaluated matched dried blood spots (DBS) and dried plasma spots (DPS) against plasma using the Abbott M 2000 (Abbott) and Roche Cobas Ampliprep/Cobas TaqMan (CAP/CTM) quantitative viral load assays in western Kenya. Matched plasma DBS and DPS were obtained from 200 HIV-1 infected antiretroviral treatment (ART)-experienced patients attending patient support centers in Western Kenya. Standard quantitative assay performance parameters with accompanying 95% confidence intervals (CI) were assessed at the assays lower detection limit (400cps/ml for CAP/CTM and 550cps/ml for Abbott) using SAS version 9.2. Receiver operating curves (ROC) were further used to assess viral-load thresholds with best assay performance (reference assay CAP/CTM plasma). Using the Abbott test, the sensitivity and specificity, respectively, for DPS were (97.3%, [95%CI: 93.2-99.2] and 98.1% [95%CI: 89.7-100]) and those for DBS (93.9% [95%CI: 88.8-97.2] and 88.0% [95%CI: 82.2-92.4]). The correlation and agreement using paired plasma and DPS/DBS were strong, with r2 = 90.5 and rc = 68.1. The Bland-Altman relative percent change was 95.3 for DPS, (95%CI: 90.4-97.7) and 73.6 (95%CI: 51.6-86.5) for DBS. Using the CAP/CTM assay, the sensitivity for DBS was significantly higher compared to DPS (100.0% [95% CI: 97.6-100.0] vs. 94.7% [95%CI: 89.8-97.7]), while the specificity for DBS was lower: 4%, [95% CI: 0.4-13.7] compared to DPS: 94.0%, [95% CI: 83.5-98.7]. When compared under different clinical relevant thresholds, the accuracy for the Abbott assay was 95% at the 1000cps/ml cut-off with a sensitivity and specificity of 96.6% [95% CI 91.8-98.7] and 90

  9. Analytical characteristics and comparative evaluation of Aptima HCV quant Dx assay with the Abbott RealTime HCV assay and Roche COBAS AmpliPrep/COBAS TaqMan HCV quantitative test v2.0.

    Science.gov (United States)

    Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A

    2017-04-04

    The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low

  10. Comparación de los métodos de cuantificación de carga viral de VIH: COBAS® AmpliPrep/COBAS® TaqMan HIV-1 test, v 2.0, y VERSANT HIV-1 RNA 1.0 Assay (kPCR Comparison of COBAS® AmpliPrep/COBAS® TaqMan HIV-1 test, v 2.0 and VERSANT HIV-1 RNA 1.0 (kPCR assays for HIV-1 plasma viral load

    Directory of Open Access Journals (Sweden)

    María Isabel Múnera-Jaramillo

    2012-03-01

    Full Text Available Objetivo. El propósito del estudio fue evaluar el desempeño del método VERSANTHIV-1RNA 1.0 Assay® (kPCR (Siemens, para la cuantificación de la carga viral en pacientes con VIH-1, en comparación con el método COBAS® AmpliPrep/COBAS TaqMan HIV-1 test®, v2.0 (Roche Diagnostics (CAP/CTM. Métodos. Las muestras fueron tomadas en dos tubos con EDTA, de 60 pacientes remitidos por el médico tratante para pruebas de carga viral como parte de su control de rutina de VIH/sida, y fueron procesadas para la cuantificación del ARN del VIH-1 por ambas técnicas. Se hizo análisis de regresión y se calcularon los coeficientes de correlación de Pearson, y los de correlación y concordancia de Lin. Se evalúo la concordancia entre las dos técnicas mediante el método de Bland-Altman. Resultados. El promedio de la carga viral por el método CAP/CTM fue 3,2±1,4 long10 copias/ml y, por el método kPCR, 3,0±1,3 long10 copias/ml. El 86,7 % de muestras presentó diferencias entre los dos métodos, menores de 0,5 long10 copias/ml, y el 13,3 % presentó diferencias mayores. El coeficiente de correlación de Pearson entre los dos métodos fue de 0,97 (IC95% 0,95-0,99 y el índice kappa ponderado entre los dos métodos en diferentes rangos de concentración, fue de 0,91 (IC95% 0,87-0,96. El promedio de las diferencias entre las mediciones fue 0,22 long10 copias/ml (IC95% -0,45 a 0,89. Conclusión. Las dos técnicas evaluadas fueron comparables, con el método kPCR se observaron resultados más bajos.Objective: The purpose of this study was to evaluate the performance of the kPCR VERSANT (™ 440 HIV-1RNA 3.0 Assay® (Siemens method for the quantification of viral load in HIV-1 patients, compared to the COBAS AmpliPrep/COBASTaqMan HIV-1 test®, v. 2.0 (Roche Diagnostics (CAP/CTM. Methods: Samples were taken in 2 tubes with EDTA, in 60 patients referred by the attending physician for viral load tests as part of their routine control of HIV/AIDS, and were

  11. TaqMan Real-Time Polymerase Chain Reaction and ...

    African Journals Online (AJOL)

    ISSN: 1596-5996 (print); 1596-9827 (electronic) ... Alcohol in humans is oxidized to acetaldehyde, which in turn is oxidized to .... cool to room temperature (15 - 25 oC) for at least. 5 min, and .... logical candidate gene for alcohol dependence. A.

  12. Performance of the cobas Hepatitis B virus (HBV) test using the cobas 4800 system and comparison of HBV DNA quantification ability between the COBAS AmpliPrep/COBAS TaqMan HBV test version 2.0 and cobas HBV test.

    Science.gov (United States)

    Shin, Kyung-Hwa; Lee, Hyun-Ji; Chang, Chulhun L; Kim, Hyung-Hoi

    2018-04-01

    Hepatitis B virus (HBV) DNA levels are used to predict the response to therapy, determine therapy initiation, monitor resistance to therapy, and establish treatment success. To verify the performance of the cobas HBV test using the cobas 4800 system for HBV DNA quantification and to compare the HBV DNA quantification ability between the cobas HBV test and COBAS AmpliPrep/COBAS TaqMan HBV version 2.0 (CAP/CTM v2.0). The precision, linearity, and limit of detection of the cobas HBV test were evaluated using the 4th World Health Organization International Standard material and plasma samples. Clinical samples that yielded quantitative results using the CAP/CTM v2.0 and cobas HBV tests were subjected to correlational analysis. Three hundred forty-nine samples were subjected to correlational analysis, among which 114 samples showed results above the lower limit of quantification. Comparable results were obtained ([cobas HBV test] = 1.038 × [CAP/CTM v2.0]-0.173, r = 0.914) in 114 samples, which yielded values above the lower limit of quantification. The results for 86.8% of the samples obtained using the cobas HBV test were within 0.5 log 10 IU/mL of the CAP/CTM v2.0 results. The total precision values against the low and high positive controls were 1.4% (mean level: 2.25 log 10 IU/mL) and 3.2% (mean level: 6.23 log 10 IU/mL), respectively. The cobas HBV test demonstrated linearity (1.15-6.75 log 10 IU/mL, y = 0.95 × 6 + 0.17, r 2  = 0.994). The cobas HBV test showed good correlation with CAP/CTM v2.0, and had good precision and an acceptable limit of detection. The cobas HBV test using the cobas 4800 is a reliable method for quantifying HBV DNA levels in the clinical setting. Copyright © 2018. Published by Elsevier B.V.

  13. Muscle segment homeobox genes direct embryonic diapause by limiting inflammation in the uterus

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Jeeyeon; Burnum-Johnson, Kristin E.; Bartos, Amanda; Li, Yingju; Baker, Erin Shammel; Tilton, Susan C.; Webb-Robertson, Bobbie-Jo M.; Piehowski, Paul D.; Monroe, Matthew E.; Jegga, Anil; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K.

    2015-06-11

    Embryonic diapause (delayed implantation) is a reproductive strategy widespread in the animal kingdom. Under this condition, embryos at the blastocyst stage become dormant simultaneously with uterine quiescence until environmental or physiological conditions are favorable for the survival of the mother and newborn. Under favorable conditions, activation of the blastocyst and uterus ensues with implantation and progression of pregnancy. Although endocrine factors are known to participate in this process, the underlying molecular mechanism coordinating this phenomenon is not clearly understood. We recently found that uterine muscle segment homeobox (Msx) transcription factors are critical for the initiation and maintenance of delayed implantation in mice. To better understand why Msx genes are critical for delayed implantation, we compared uterine proteomics profiles between littermate floxed (Msx1/Msx2f/f) mice and mice with uterine deletion of Msx genes (Msx1/Msx2d/d) under delayed conditions. In Msx1/Msx2d/d uteri, pathways including protein translation, ubiquitin-proteasome system, inflammation, chaperone-mediated protein folding, and endoplasmic reticulum (ER) stress were enriched, and computational modeling showed intersection of these pathways on inflammatory responses. Indeed, increases in the ubiquitin-proteasome system and inflammation conformed to proteotoxic and ER stress in Msx1/Msx2d/d uteri under delayed conditions. Interestingly, treatment with a proteasome inhibitor bortezomib further exacerbated ER stress in Msx1/Msx2d/d uteri with aggravated inflammatory response, deteriorating rate of blastocyst recovery and failure to sustain delayed implantation. This study highlights a previously unrecognized role for Msx in preventing proteotoxic stress and inflammatory responses to coordinate embryo dormancy and uterine quiescence during embryonic diapause.

  14. Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus*

    Science.gov (United States)

    Cha, Jeeyeon; Burnum-Johnson, Kristin E.; Bartos, Amanda; Li, Yingju; Baker, Erin S.; Tilton, Susan C.; Webb-Robertson, Bobbie-Jo M.; Piehowski, Paul D.; Monroe, Matthew E.; Jegga, Anil G.; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K.

    2015-01-01

    Embryonic diapause is a reproductive strategy widespread in the animal kingdom. This phenomenon is defined by a temporary arrest in blastocyst growth and metabolic activity within a quiescent uterus without implantation until the environmental and maternal milieu become favorable for pregnancy to progress. We found that uterine Msx expression persists during diapause across species; their inactivation in the mouse uterus results in termination of diapause with the development of implantation-like responses (“pseudoimplantation”) that ultimately succumbed to resorption. To understand the cause of this failure, we compared proteome profiles between floxed and Msx-deleted uteri. In deleted uteri, several functional networks, including transcription/translation, ubiquitin-proteasome, inflammation, and endoplasmic reticulum stress, were dysregulated. Computational modeling predicted intersection of these pathways on an enhanced inflammatory signature. Further studies showed that this signature was reflected in increased phosphorylated IκB levels and nuclear NFκB in deleted uteri. This was associated with enhanced proteasome activity and endoplasmic reticulum stress. Interestingly, treatment with anti-inflammatory glucocorticoid (dexamethasone) reduced the inflammatory signature with improvement of the diapause phenotype. These findings highlight an unexpected role of uterine Msx in limiting aberrant inflammatory responses to maintain embryonic diapause. PMID:25931120

  15. Muscle Segment Homeobox Genes Direct Embryonic Diapause by Limiting Inflammation in the Uterus.

    Science.gov (United States)

    Cha, Jeeyeon; Burnum-Johnson, Kristin E; Bartos, Amanda; Li, Yingju; Baker, Erin S; Tilton, Susan C; Webb-Robertson, Bobbie-Jo M; Piehowski, Paul D; Monroe, Matthew E; Jegga, Anil G; Murata, Shigeo; Hirota, Yasushi; Dey, Sudhansu K

    2015-06-12

    Embryonic diapause is a reproductive strategy widespread in the animal kingdom. This phenomenon is defined by a temporary arrest in blastocyst growth and metabolic activity within a quiescent uterus without implantation until the environmental and maternal milieu become favorable for pregnancy to progress. We found that uterine Msx expression persists during diapause across species; their inactivation in the mouse uterus results in termination of diapause with the development of implantation-like responses ("pseudoimplantation") that ultimately succumbed to resorption. To understand the cause of this failure, we compared proteome profiles between floxed and Msx-deleted uteri. In deleted uteri, several functional networks, including transcription/translation, ubiquitin-proteasome, inflammation, and endoplasmic reticulum stress, were dysregulated. Computational modeling predicted intersection of these pathways on an enhanced inflammatory signature. Further studies showed that this signature was reflected in increased phosphorylated IκB levels and nuclear NFκB in deleted uteri. This was associated with enhanced proteasome activity and endoplasmic reticulum stress. Interestingly, treatment with anti-inflammatory glucocorticoid (dexamethasone) reduced the inflammatory signature with improvement of the diapause phenotype. These findings highlight an unexpected role of uterine Msx in limiting aberrant inflammatory responses to maintain embryonic diapause. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Molecular Cloning of Human Gene(s) Directing the Synthesis of Nervous System Cholinesterases

    Science.gov (United States)

    1987-09-01

    responsible for at least some of the symptoms observed in hyperthyroidism . The location of primary sequences of human ChE showing high homology to bovine...verteLrates both classes exist in the heart. Humans having non- functional BuChE in the serum show no known symptoms of illness (15) and exhibit normal...become a "physiological nuisance" in cases of hyperthyroidism (such as Graves’ ophthalmopathy). In this disorder, there exists an overproduction of the

  17. Cellular zinc fluxes and the regulation of apoptosis/gene-directed cell death.

    Science.gov (United States)

    Truong-Tran, A Q; Ho, L H; Chai, F; Zalewski, P D

    2000-05-01

    The maintenance of discrete subcellular pools of zinc (Zn) is critical for the functional and structural integrity of cells. Among the important biological processes influenced by Zn is apoptosis, a process that is important in cellular homeostasis (an important cellular homeostatic process). It has also been identified as a major mechanism contributing to cell death in response to toxins and in disease, offering hope that novel therapies that target apoptotic pathways may be developed. Because Zn levels in the body can be increased in a relatively nontoxic manner, it may be possible to prevent or ameliorate degenerative disorders that are associated with high rates of apoptotic cell death. This review begins with brief introductions that address, first, the cellular biology of Zn, especially the critical labile Zn pools, and, second, the phenomenon of apoptosis. We then review the evidence relating Zn to apoptosis and address three major hypotheses: (1) that a specific pool or pools of intracellular labile Zn regulates apoptosis; (2) that systemic changes in Zn levels in the body, due to dietary factors, altered physiological states or disease, can influence cell susceptibility to apoptosis, and (3) that this altered susceptibility to apoptosis contributes to pathophysiological changes in the body. Other key issues are the identity of the molecular targets of Zn in the apoptotic cascade, the types of cells and tissues most susceptible to Zn-regulated apoptosis, the role of Zn as a coordinate regulator of mitosis and apoptosis and the apparent release of tightly bound intracellular pools of Zn during the later stages of apoptosis. This review concludes with a section highlighting areas of priority for future studies.

  18. Rapid approach for cloning bacterial single-genes directly from soils ...

    African Journals Online (AJOL)

    Obtaining functional genes of bacteria from environmental samples usually depends on library-based approach which is not favored as its large amount of work with small possibility of positive clones. A kind of bacterial single-gene encoding glutamine synthetase (GS) was selected as example to detect the efficiency of ...

  19. Specificity of a novel TaqMan PCR method for detection of poultry DNA

    NARCIS (Netherlands)

    Scholtens-Toma, Ingrid; Prins, Theo W.; Raamsdonk, Van Leo W.D.

    2017-01-01

    After the Bovine Spongiform Encephalopathy (BSE) crisis emerged in 1985/1986, all processed animal proteins (PAPs) were finally banned for use in animal feed in the European Union. To partially lift this feed ban, paths for re-introduction of PAPs from species other than ruminants e.g. pig and

  20. DETECTION AND IDENTIFICATION OF PATHOGENIC CANDIDA SPECIES IN WATER USING FLOW CYTOMETRY COUPLED WITH TAQMAN PCR

    Science.gov (United States)

    As the incidence of human fungal infection increases, the ability to detect and identify pathogenic fungi in potential environmental reservoirs becomes increasingly important for disease control. PCR based assays are widely used for diagnostic purposes, but may be inadequate for...

  1. Semiautomated TaqMan PCR screening of GMO labelled samples for (unauthorised) GMOs.

    Science.gov (United States)

    Scholtens, Ingrid M J; Molenaar, Bonnie; van Hoof, Richard A; Zaaijer, Stephanie; Prins, Theo W; Kok, Esther J

    2017-06-01

    In most countries, systems are in place to analyse food products for the potential presence of genetically modified organisms (GMOs), to enforce labelling requirements and to screen for the potential presence of unauthorised GMOs. With the growing number of GMOs on the world market, a larger diversity of methods is required for informative analyses. In this paper, the specificity of an extended screening set consisting of 32 screening methods to identify different crop species (endogenous genes) and GMO elements was verified against 59 different GMO reference materials. In addition, a cost- and time-efficient strategy for DNA isolation, screening and identification is presented. A module for semiautomated analysis of the screening results and planning of subsequent event-specific tests for identification has been developed. The Excel-based module contains information on the experimentally verified specificity of the element methods and of the EU authorisation status of the GMO events. If a detected GMO element cannot be explained by any of the events as identified in the same sample, this may indicate the presence of an unknown unauthorised GMO that may not yet have been assessed for its safety for humans, animals or the environment.

  2. Semiautomated TaqMan PCR screening of GMO labelled samples for (unauthorised) GMOs

    NARCIS (Netherlands)

    Scholtens-Toma, Ingrid; Molenaar, Bonnie; Hoof, van Richard A.; Zaaijer, Stephanie; Prins, Theo W.; Kok, Esther J.

    2017-01-01

    In most countries, systems are in place to analyse food products for the potential presence of genetically modified organisms (GMOs), to enforce labelling requirements and to screen for the potential presence of unauthorised GMOs. With the growing number of GMOs on the world market, a larger

  3. Application of rapid onsite PCR (TaqMan) for Phytophthora ramorum under U.S. conditions

    Science.gov (United States)

    Kelvin Hughes; Jenny Tomlinson; Neil Boonham; Kelly Ivors; Matteo Garbelotto; Ian Barker

    2006-01-01

    Currently, diagnosis of Phytophthora ramorum involves sending samples to a laboratory for traditional isolation and morphological characterisation, and/or PCR analysis. This can take as long as 2 weeks from sampling to final diagnosis. However, the Plant Health Group, Central Science Laboratory, has produced on-site DNA extraction and real-time PCR (...

  4. Investigations into the biodegradation of microcystin-LR in wastewaters

    International Nuclear Information System (INIS)

    Ho, Lionel; Hoefel, Daniel; Palazot, Sebastien; Sawade, Emma; Newcombe, Gayle; Saint, Christopher P.; Brookes, Justin D.

    2010-01-01

    Microcystins are potent hepatotoxins that can be produced by cyanobacteria. These organisms can proliferate in wastewaters due to a number of factors including high concentrations of nutrients for growth. As treated wastewaters are now being considered as supplementary drinking water sources, in addition to their frequent use for irrigated agriculture, it is imperative that these wastewaters are free of toxins such as microcystins. This study investigated the potential for biodegradation of microcystin-LR (MCLR) in wastewaters through a biological sand filtration experiment and in static batch reactor experiments. MCLR was effectively removed at a range of concentrations and at various temperatures, with degradation attributed to the action of microorganisms indigenous to the wastewaters. No hepatotoxic by-products were detected following the degradation of MCLR as determined by a protein phosphatase inhibition assay. Using TaqMan polymerase chain reaction, the first gene involved in bacterial degradation of MCLR (mlrA) was detected and the responsible bacteria shown to increase with the amount of MCLR being degraded. This finding suggested that the degradation of MCLR was dependent upon the abundance of MCLR-degrading organisms present within the wastewater, and that MCLR may provide bacteria with a significant carbon source for proliferation; in turn increasing MCLR removal.

  5. A xylanase gene directly cloned from the genomic DNA of alkaline wastewater sludge showing application potential in the paper industry.

    Science.gov (United States)

    Zhao, Yanyu; Luo, Huiying; Meng, Kun; Shi, Pengjun; Wang, Guozeng; Yang, Peilong; Yuan, Tiezheng; Yao, Bin

    2011-09-01

    A xylanase gene, aws-2x, was directly cloned from the genomic DNA of the alkaline wastewater sludge using degenerated PCR and modified TAIL-PCR. The deduced amino acid sequence of AWS-2x shared the highest identity (60%) with the xylanase from Chryseobacterium gleum belonging to the glycosyl hydrolase GH family 10. Recombinant AWS-2x was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 7.5 and 55 °C, maintained more than 50% of maximal activity when assayed at pH 9.0, and was stable over a wide pH range from 4.0 to 11.0. The specific activity of AWS-2x towards hardwood xylan (beechwood and birchwood xylan) was significantly higher than that to cereal xylan (oat spelt xylan and wheat arabinoxylan). These properties make AWS-2x a potential candidate for application in the pulp and paper industry.

  6. A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

    OpenAIRE

    Uozumi, N; Sakurai, K; Sasaki, T; Takekawa, S; Yamagata, H; Tsukagoshi, N; Udaka, S

    1989-01-01

    The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other a...

  7. Genome-wide identification of genes directly regulated by the pleiotropic transcription factor Spx in Bacillus subtilis

    Czech Academy of Sciences Publication Activity Database

    Rochat, T.; Nicolas, P.; Delumeau, O.; Rabatinová, Alžběta; Korelusová, Jana; Leduc, A.; Bessieres, P.; Dervyn, E.; Krásný, Libor; Noirot, P.

    2012-01-01

    Roč. 40, č. 19 (2012), s. 9571-9583 ISSN 0305-1048 R&D Projects: GA ČR GAP302/11/0855 Institutional support: RVO:61388971 Key words : C-TERMINAL DOMAIN * RNA-POLYMERASE * DISULFIDE STRESS Subject RIV: CE - Biochemistry Impact factor: 8.278, year: 2012

  8. Intratumor genetic heterogeneity of breast carcinomas as determined by fine needle aspiration and TaqMan low density array

    DEFF Research Database (Denmark)

    Lyng, Maria B.; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

    2007-01-01

    BACKGROUND: Gene expression profiling is thought to be an important tool in determining treatment strategies for breast cancer patients. Tissues for such analysis may at a preoperative stage be obtained, by fine needle aspiration (FNA) allowing initiation of neoadjuvant treatment. To evaluate the...... of breast carcinomas, as sampled by FNA, does not prohibit generation of useful gene profiles for treatment decision making. However, sampling and analysis strategies should take heterogeneity within a tumor, and varying heterogeneity amongst the single genes, into account...

  9. A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2).

    Science.gov (United States)

    Duarte, Margarida Dias; Carvalho, Carina L; Barros, Silvia C; Henriques, Ana M; Ramos, Fernanda; Fagulha, Teresa; Luís, Tiago; Duarte, Elsa L; Fevereiro, Miguel

    2015-07-01

    A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3' end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R(2) value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

    DEFF Research Database (Denmark)

    Stilwell, Natalie K.; Whittington, Richard J.; Hick, Paul M.

    2018-01-01

    Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ran...

  11. Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

    Directory of Open Access Journals (Sweden)

    Henrich Birgit

    2008-04-01

    Full Text Available Abstract Background Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men. Results The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1 -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male. Conclusion This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies.

  12. Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

    Science.gov (United States)

    Schaeffer, Anke; Henrich, Birgit

    2008-01-01

    Background Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men. Results The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1) -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H) -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male. Conclusion This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies. PMID:18447917

  13. Optimization of a 12-hour TaqMan PCR-based method for detection of Salmonella bacteria in meat

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Krause, Michael; Hansen, F.

    2007-01-01

    no positive effects and resulted in decreased reproducibility. Increasing the amount of PCR template DNA from 5 to 20 mu l improved the threshold cycle value by approximately 2. The improved 12-h PCR method was successfully compared to a reference culture method with 100 minced meat and poultry samples...... the highest number of salmonellae. When analyzing minced meat samples, positive effects of increasing the initial sampling volume from 1 to 5 ml and increasing the amount of paramagnetic particles to 90 mu l were observed. However, washing the pellet and eluting the DNA in reduced volumes (25 and 50 mu l) had...

  14. Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays

    DEFF Research Database (Denmark)

    Engle, Ronald E; Russell, Rodney S; Purcell, Robert H

    2008-01-01

    A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first...

  15. Molecular detection of Phytophthora ramorum by real-time PCR using Taqman, SYBR Green and molecular beacons with three genes

    Science.gov (United States)

    G.J. Bilodeau; C.A. Lévesque; A.W.A.M. De Cock; C. Duchaine; G. Kristjansson; R.C. Hamelin

    2006-01-01

    Sudden oak death, caused by Phytophthora ramorum, is a severe disease that can affect numerous species of trees and shrubs. This pathogen has been spread via nursery stock, and quarantine measures are currently in place to prevent further spread. Molecular assays have been developed to rapidly detect and identify P. ramorum, but...

  16. Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay.

    Science.gov (United States)

    Corey, Lawrence; Huang, Meei-Li; Selke, Stacy; Wald, Anna

    2005-07-01

    While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques. Copyright (c) 2005 Wiley-Liss, Inc.

  17. 17α-Ethinylestradiol (EE2) treatment of wild roach (Rutilus rutilus) during early life development disrupts expression of genes directly involved in the feedback cycle of estrogen.

    Science.gov (United States)

    Nikoleris, Lina; Hultin, Cecilia L; Hallgren, Per; Hansson, Maria C

    2016-02-01

    Fish are more sensitive to introduced disturbances from synthetic endocrine disrupting compounds during early life phases compared with mature stages. 17α-Ethinylestradiol (EE2), which is the active compound in human oral contraceptives and hormone replacement therapies, is today ever present in the effluents from sewage treatment plants. EE2 targets and interacts with the endogenous biological systems of exposed vertebrates resulting in to large extents unknown short- and long-term effects. We investigated how EE2 exposure affects expression profiles of a large number of target genes during early life of roach (Rutilus rutilus). We exposed fertilized roach eggs collected from a lake in Southern Sweden to EE2 for 12weeks together with 1+-year-old roach in aquaria. We measured the gene expression of the estrogen receptor (esr)1/2a/2b, androgen receptor (ar), vitellogenin, cytochrome P450 (cyp)19a1a/1b in fertilized eggs; newly hatched larvae; 12-week-old fry; and juvenile wild roach (1+-year-old). Results shows that an EE2 concentration as low as 0.5ng/L significantly affects gene expression during early development. Gene expression responses vary both among life stages and molecular receptors. We also show that the gene profile of the estrogen feedback cycle to a large extent depends on the relationship between the three esr genes and the two cyp19a1 genes, which are all up-regulated with age. Results indicate that a disruption of the natural activity of the dominant esr gene could lead to detrimental biological effects if EE2 exposure occurs during development, even if this exposure occurred for only a short period. Copyright © 2015. Published by Elsevier Inc.

  18. Ideal crop plant architecture is mediated by tassels replace upper ears1, a BTB/POZ ankyrin repeat gene directly targeted by TEOSINTE BRANCHED1.

    Science.gov (United States)

    Dong, Zhaobin; Li, Wei; Unger-Wallace, Erica; Yang, Jinliang; Vollbrecht, Erik; Chuck, George

    2017-10-10

    Axillary branch suppression is a favorable trait bred into many domesticated crop plants including maize compared with its highly branched wild ancestor teosinte. Branch suppression in maize was achieved through selection of a gain of function allele of the teosinte branched1 (tb1) transcription factor that acts as a repressor of axillary bud growth. Previous work indicated that other loci may function epistatically with tb1 and may be responsible for some of its phenotypic effects. Here, we show that tb1 mediates axillary branch suppression through direct activation of the tassels replace upper ears1 ( tru1 ) gene that encodes an ankyrin repeat domain protein containing a BTB/POZ motif necessary for protein-protein interactions. The expression of TRU1 and TB1 overlap in axillary buds, and TB1 binds to two locations in the tru1 gene as shown by chromatin immunoprecipitation and gel shifts. In addition, nucleotide diversity surveys indicate that tru1 , like tb1 , was a target of selection. In modern maize, TRU1 is highly expressed in the leaf trace vasculature of axillary internodes, while in teosinte, this expression is highly reduced or absent. This increase in TRU1 expression levels in modern maize is supported by comparisons of relative protein levels with teosinte as well as by quantitative measurements of mRNA levels. Hence, a major innovation in creating ideal maize plant architecture originated from ectopic overexpression of tru1 in axillary branches, a critical step in mediating the effects of domestication by tb1.

  19. Adenoviral delivery of pan-caspase inhibitor p35 enhances bystander killing by P450 gene-directed enzyme prodrug therapy using cyclophosphamide+

    International Nuclear Information System (INIS)

    Doloff, Joshua C; Su, Ting; Waxman, David J

    2010-01-01

    Cytochrome P450-based suicide gene therapy for cancer using prodrugs such as cyclophosphamide (CPA) increases anti-tumor activity, both directly and via a bystander killing mechanism. Bystander cell killing is essential for the clinical success of this treatment strategy, given the difficulty of achieving 100% efficient gene delivery in vivo using current technologies. Previous studies have shown that the pan-caspase inhibitor p35 significantly increases CPA-induced bystander killing by tumor cells that stably express P450 enzyme CYP2B6 (Schwartz et al, (2002) Cancer Res. 62: 6928-37). To further develop this approach, we constructed and characterized a replication-defective adenovirus, Adeno-2B6/p35, which expresses p35 in combination with CYP2B6 and its electron transfer partner, P450 reductase. The expression of p35 in Adeno-2B6/p35-infected tumor cells inhibited caspase activation, delaying the death of the CYP2B6 'factory' cells that produce active CPA metabolites, and increased bystander tumor cell killing compared to that achieved in the absence of p35. Tumor cells infected with Adeno-2B6/p35 were readily killed by cisplatin and doxorubicin, indicating that p35 expression is not associated with acquisition of general drug resistance. Finally, p35 did not inhibit viral release when the replication-competent adenovirus ONYX-017 was used as a helper virus to facilitate co-replication and spread of Adeno-2B6/p35 and further increase CPA-induced bystander cell killing. The introduction of p35 into gene therapeutic regimens constitutes an effective approach to increase bystander killing by cytochrome P450 gene therapy. This strategy may also be used to enhance other bystander cytotoxic therapies, including those involving the production of tumor cell toxic protein products

  20. A locked nucleic acid (LNA-based real-time PCR assay for the rapid detection of multiple bacterial antibiotic resistance genes directly from positive blood culture.

    Directory of Open Access Journals (Sweden)

    Lingxiang Zhu

    Full Text Available Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA-based quantitative real-time PCR (LNA-qPCR method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1-10 colony forming units (CFU per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4% were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.

  1. Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

    Science.gov (United States)

    Harvey, John J; Chester, Stephanie; Burke, Stephen A; Ansbro, Marisela; Aden, Tricia; Gose, Remedios; Sciulli, Rebecca; Bai, Jing; DesJardin, Lucy; Benfer, Jeffrey L; Hall, Joshua; Smole, Sandra; Doan, Kimberly; Popowich, Michael D; St George, Kirsten; Quinlan, Tammy; Halse, Tanya A; Li, Zhen; Pérez-Osorio, Ailyn C; Glover, William A; Russell, Denny; Reisdorf, Erik; Whyte, Thomas; Whitaker, Brett; Hatcher, Cynthia; Srinivasan, Velusamy; Tatti, Kathleen; Tondella, Maria Lucia; Wang, Xin; Winchell, Jonas M; Mayer, Leonard W; Jernigan, Daniel; Mawle, Alison C

    2016-02-01

    In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT. When compared to CDC LDTs, TAC assays were generally one to two logs less sensitive depending on the site performing the analysis. Finally, TAC assays were generally more sensitive than their counterparts in three different commercial multi-respiratory panels. TAC technology allows users to spot customized assays and design TAC layout, simplify assay setup, conserve specimen, dramatically reduce contamination potential, and as demonstrated in this study, analyze multiple samples in parallel with good reproducibility between instruments and operators. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Interlaboratory validation of an improved U.S. Food and Drug Administration method for detection of Cyclospora cayetanensis in produce using TaqMan real-time PCR

    Science.gov (United States)

    A collaborative validation study was performed to evaluate the performance of a new U.S. Food and Drug Administration method developed for detection of the protozoan parasite, Cyclospora cayetanensis, on cilantro and raspberries. The method includes a sample preparation step in which oocysts are re...

  3. Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan probe and SYBR green real-time PCR assays

    Science.gov (United States)

    The ectoparasitic stubby root nematode Paratrichodorus allius transmits Tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. This study developed a diagnostic method for direct quantification of P. allius from soil DNA using a Taq...

  4. Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics

    NARCIS (Netherlands)

    Molenkamp, Richard; van der Ham, Alwin; Schinkel, Janke; Beld, Marcel

    2007-01-01

    One of the most interesting aspects of real-time PCR based on the detection of fluorophoric labeled oligonucleotides is the possibility of being able to detect conveniently multiple targets in the same PCR reaction. Recently, Roche Diagnostics launched a real-time PCR platform, the LightCycler480

  5. Detection and identification of six Monilinia spp. causing brown rot using TaqMan real-time PCR from pure cultures and infected apple fruit

    Science.gov (United States)

    Brown rot is a severe disease affecting stone and pome fruits. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, Monilinia laxa, Monilinia fructigena, Monilia polystroma, Monilia mumecola and Monilia yunnanensis. Because of differenc...

  6. Development of a taqman-based real-time PCR assay for the rapid and specific detection of novel duck- origin goose parvovirus.

    Science.gov (United States)

    Wang, Jianchang; Wang, Jinfeng; Cui, Yuan; Nan, Huizhu; Yuan, Wanzhe

    2017-08-01

    A real-time PCR assay was developed for specific detection of novel duck-origin goose parvovirus (N-GPV), the etiological agent of duck beak atrophy and dwarfism syndrome (BADS). The detection limit of the assay was 10 2 copies. The assay was useful in the prevention and control of BADS. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Quantification of ochratoxin A-producing molds in food products by SYBR Green and TaqMan real-time PCR methods

    DEFF Research Database (Denmark)

    Rodríguez, Alicia; Rodríguez, Mar; Luque, M. Isabel

    2011-01-01

    , usually reported in food products, were used as references. All strains were tested for OTA production by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). The ability of the optimized qPCR protocols to quantify OTA......Ochratoxin A (OTA) is a mycotoxin synthesized by a variety of different fungi, most of them from the genera Penicillium and Aspergillus. Early detection and quantification of OTA producing species is crucial to improve food safety. In the present work, two protocols of real-time qPCR based on SYBR......-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1 x 104 to 10 conidia/g per reaction for all qPCR assays in the different food matrices (cooked and cured products and fruits). The detection limit in all inoculated foods ranged between...

  8. Gebruik van TaqMan PCR voor het kwantificeren van Fusarium spp. en Microdochium nivale in gewassen en gewasresten van tarwe

    NARCIS (Netherlands)

    Köhl, J.; Haas, de B.H.; Kastelein, P.; Burgers, S.L.G.E.; Waalwijk, C.

    2005-01-01

    Samenvattingen van 5 presentaties: 'Heterogenity of Dutch Fusarium oxysporum strains isolated as forma specialis radicis-lycopersici';'Een proteomics benadering om eiwitten te identificeren die door Fusarium oxysporum worden uitgescheiden in xyleemsap van tomaat'; 'Ontwikkeling en implementatie van

  9. Gebruik van TaqMan PCR voor het kwantificeren van Fusarium spp. en Microdochium nivale in gewassen en gewasresten van tarwe.

    NARCIS (Netherlands)

    Köhl, J.; Haas, de B.H.; Kastelein, P.; Burgers, S.L.G.E.; Waalwijk, C.

    2005-01-01

    Infecties van tarwe door toxigene Fusarium spp. kunnen leiden tot contaminatie van graan met diverse mycotoxines. Voorkoming van mycotoxinebesmetting is uit oogpunt van voedselveiligheid, diergezondheid, maar ook bedrijfseconomisch, vereist. Voor vier Fusariumsoorten is een kwantitatieve detectie

  10. Comparative, Collaborative, and On-Site Validation of a TaqMan PCR Method as a Tool for Certified Production of Fresh, Campylobacter-Free Chickens

    DEFF Research Database (Denmark)

    Krause, Michael; Josefsen, Mathilde Hartmann; Lund, Marianne

    2006-01-01

    , a faster, real-time PCR approach was validated in comparative and collaborative trials, based on recommendations from the Nordic system for validation of alternative microbiological methods (NordVal). The comparative real-time PCR trial was performed in comparison to two reference culture protocols...... fulfilled the NordVal validation criteria and has since been implemented at a major abattoir....

  11. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Science.gov (United States)

    Wadhwa, Ashutosh; Wilkins, Kimberly; Gao, Jinxin; Condori Condori, Rene Edgar; Gigante, Crystal M; Zhao, Hui; Ma, Xiaoyue; Ellison, James A; Greenberg, Lauren; Velasco-Villa, Andres; Orciari, Lillian; Li, Yu

    2017-01-01

    Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

  12. A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses.

    Directory of Open Access Journals (Sweden)

    Ashutosh Wadhwa

    2017-01-01

    Full Text Available Rabies, resulting from infection by Rabies virus (RABV and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

  13. Monkeypox detection in rodents using real-time 3'minor groove binder Taqman assays on the Roche LightCycler, Laboratory Investigation 84:1200 - 1208

    National Research Council Canada - National Science Library

    Kulesh, David

    2004-01-01

    .... The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture...

  14. Novel TaqMan PCR screening methods for element cry3A and construct gat/T-pinII to support detection of both known and unknown GMOs

    NARCIS (Netherlands)

    Prins, Theo W.; Hoof, van Richard A.; Scholtens, Ingrid M.J.; Kok, Esther J.

    2017-01-01

    The import and use of genetically modified organisms (GMOs) is strictly regulated in the European Union. In order to maintain the legislation on GMOs, a genetic element screening is generally applied as a first step to detect authorised as well as unauthorised GMOs. Subsequent identification of

  15. Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab

    Science.gov (United States)

    Powdery scab caused by the protist Spongospora subterranea and common scab caused by pathogenic bacteria in the genus Streptomyces are two important potato diseases worldwide. The symptoms of the two diseases are often similar, and diagnosis of the two diseases is therefore challenging for potato r...

  16. Development of duplex real-time RT-PCR based on Taqman technology for detecting simultaneously the genome of pan-enterovirus and enterovirus 71.

    Science.gov (United States)

    Hwang, Seoyeon; Kang, Byunghak; Hong, Jiyoung; Kim, Ahyoun; Kim, Hyejin; Kim, Kisang; Cheon, Doo-Sung

    2013-07-01

    Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks. Copyright © 2013 Wiley Periodicals, Inc.

  17. Comparison of one commercial and two in-house TaqMan multiplex real-time PCR assays for detection of enteropathogenic, enterotoxigenic and enteroaggregative Escherichia coli.

    Science.gov (United States)

    Hahn, Andreas; Luetgehetmann, Marc; Landt, Olfert; Schwarz, Norbert Georg; Frickmann, Hagen

    2017-11-01

    Enteropathogenic, enterotoxigenic and enteroaggregative Escherichia coli (EPEC, ETEC, EAEC) are among the most frequent causes of diarrhoea during travel or on military deployments. Cost-efficient and reliable real-time multiplex PCR (mPCR) assays are desirable for surveillance or point prevalence studies in remote and resource-limited tropical settings. We compared one commercial PCR kit and two in-house assays without using a gold standard to estimate sensitivity and specificity of each assay. Residual materials from nucleic acid extractions of stool samples from two groups with presumably different prevalences and increased likelihood of being infected or colonised by diarrhoeagenic E. coli were included in the assessment. One group comprised samples from returnees from tropical deployments, the second group was of migrants and study participants from high-endemicity settings. Each sample was assessed with all of the PCR assays. Cycle threshold (Ct) values were descriptively compared. The calculated sensitivities for the commercial test vs. the in-house tests were for EPEC 0.84 vs. 0.89 and 0.96, for ETEC 0.83 vs. 0.76 and 0.61, and for EAEC 0.69 vs. 0.54 and 0.69. False positive results were rare - specificity was 0.94 and 0.97 for two EPEC tests and 1.0 for all other tests. Most positive samples had late Ct values corresponding to low quantities of pathogens. Discordant test results were associated with late Ct values. As commercial and in-house assays showed comparable results, in-house tests can be assumed to be safe while affording considerable savings, making them a valuable alternative for surveillance testing in resource-limited tropical areas. © 2017 John Wiley & Sons Ltd.

  18. Enhanced reliability and accuracy for field deployable bioforensic detection and discrimination of Xylella fastidiosa subsp. pauca, causal agent of citrus variegated chlorosis using razor ex technology and TaqMan quantitative PCR.

    Science.gov (United States)

    Ouyang, Ping; Arif, Mohammad; Fletcher, Jacqueline; Melcher, Ulrich; Ochoa Corona, Francisco Manuel

    2013-01-01

    A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.

  19. Genital herpes evaluation by quantitative TaqMan PCR: correlating single detection and quantity of HSV-2 DNA in cervicovaginal lavage fluids with cross-sectional and longitudinal clinical data

    Directory of Open Access Journals (Sweden)

    Cox Christopher

    2010-11-01

    Full Text Available Abstract Objective To evaluate the utility of a single quantitative PCR (qPCR measurement of HSV (HSV-1&2 DNA in cervicovaginal lavage (CVL specimens collected from women with predominantly chronic HSV-2 infection in assessing genital HSV shedding and the clinical course of genital herpes (GH within a cohort with semiannual schedule of follow up and collection of specimens. Methods Two previously described methods used for detection of HSV DNA in mucocutaneous swab samples were adapted for quantification of HSV DNA in CVLs. Single CVL specimens from 509 women were tested. Presence and quantity of CVL HSV DNA were explored in relation to observed cross-sectional and longitudinal clinical data. Results The PCR assay was sensitive and reproducible with a limit of quantification of ~50 copies per milliliter of CVL. Overall, 7% of the samples were positive for HSV-2 DNA with median log10 HSV-2 DNA copy number of 3.9 (IQR: 2.6-5.7. No HSV-1 was detected. Presence and quantity of HSV-2 DNA in CVL directly correlated with the clinical signs and symptoms of presence of active symptomatic disease with frequent recurrences. Conclusion Single qPCR measurement of HSV DNA in CVL fluids of women with chronic HSV-2 infection provided useful information for assessing GH in the setting of infrequent sampling of specimens. Observed positive correlation of the presence and quantity of HSV-2 DNA with the presence of active and more severe course of HSV-2 infection may have clinical significance in the evaluation and management of HSV-2 infected patients.

  20. Smallpox and pan-Orthodox Virus Detection by Real-Time 3’-Minor Groove Binder TaqMan Assays Oil the Roche LightCycler and the Cepheid Smart Cycler Platforms

    Science.gov (United States)

    2003-11-08

    Bacillus anthracis BA0068 Ames Sterne SPS 97.13.213 Bacillus cereus Bacillus coagulans Bacillus licheniformis Bacillus macerans Bacillus ...megaterium Bacillus polymyxa Bacillus sphaericus Bacillus stearothermophilus Bacillus subtilis subsp. niger Bacillus thuringiensis Bacillus popilliae...varicella- zoster virus, and Bacillus anthracis DNA by LightCycler polymerase chain reaction after autoclaving:

  1. Establishing diagnostic cut-off criteria for the COBAS AmpliPrep/COBAS TaqMan HIV-1 Qualitative test through validation against the Amplicor DNA test v1.5 for infant diagnosis using dried blood spots.

    Science.gov (United States)

    Maritz, Jean; Preiser, Wolfgang; van Zyl, Gert U

    2012-02-01

    As antibody testing cannot confirm HIV-1 infection in children less than 18 months of age, diagnosis in these children depends on nucleic acid testing. The COBAS(®) AmpliPrep/COBAS(®) TaqMan(®) (CAP/CTM, Roche(®) Molecular Systems, Inc., Branchburg, NJ) HIV-1 Qualitative test is a total nucleic acid real-time PCR assay utilising whole EDTA blood or dried blood spots (DBS), which recently replaced the Roche(®) AMPLICOR(®) DNA test v1.5 (Amplicor) as the diagnostic HIV PCR assay in many South African laboratories. For the Amplicor assay, stringent diagnostic criteria were previously formulated for the local population, and a comparison reported the CAP/CTM's sensitivity at 99.7% and specificity at 100% for both sample types compared to these Amplicor criteria. To validate the assay prior to introduction in our laboratory and to define stringent diagnostic cut-off criteria. Whole EDTA blood samples from patients younger than 18 months sent for routine HIV-1 diagnosis were tested by Amplicor, and positive results were confirmed from DBS. CAP/CTM assays were subsequently performed from DBS. The CAP/CTM had a sensitivity of 98.8% and a specificity of 97.1%, but a positive predictive value (PPV) of only 78.7% compared to the Amplicor assay. Samples positive by CAP/CTM but negative by Amplicor displayed poor amplification curves compared to concordant positive samples. Upon re-testing those with sufficient material available by CAP/CTM, all showed negative results. The decreased PPV may either be due to false positive CAP/CTM results, or increased sensitivity compared to the Amplicor assay. Criteria were formulated for defining presumed false-positive results. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Analytical performance of the Hologic Aptima HBV Quant Assay and the COBAS Ampliprep/COBAS TaqMan HBV test v2.0 for the quantification of HBV DNA in plasma samples

    DEFF Research Database (Denmark)

    Schønning, Kristian; Johansen, Kim; Nielsen, Lone Gilmor

    2018-01-01

    than in CAPCTMv2. Only minor differences were observed between genotype A (N = 4; average difference -0.01 Log IU/mL), B (N = 8; -0.13 Log IU/mL), C (N = 8; -0.31 Log IU/mL), D (N = 25; -0.22 Log IU/mL), and E (N = 7; -0.03 Log IU/mL). Deming regression showed that the two tests were excellently...

  3. Comparison of the Hologic Aptima HIV-1 Quant Dx Assay to the Roche COBAS Ampliprep/COBAS TaqMan HIV-1 Test v2.0 for the quantification of HIV-1 RNA in plasma samples

    DEFF Research Database (Denmark)

    Schønning, Kristian; Johansen, Kim; Landt, Bodil

    2017-01-01

    load compared to the CAPCTMv2 was observed in Bland-Altman analysis, by Deming regression (Slope 1.13) and in dilution series of clinical samples. Precision evaluated using the Acrometrix Positive Controls was similar for the High Control (CV: 1.2% vs. 1.3%; Aptima assay vs. CAPCTMv2 test, respectively...

  4. A Review of Land and Stream Classifications in Support of Developing a National Ordinary High Water Mark (OHWM) Classification

    Science.gov (United States)

    2014-08-01

    LWD Large, Woody Debris MLRA Major Land Resource Area NCDWQ North Carolina Department of Water Quality NRCS Natural Resources Conservation Service...Sinuosity, bedforms (dunes, ripples, bars), banks Bedforms (bars, pools), grains, LWD ,* sinuosity, banks Grains, banks Bedforms...steps, pools), grains, LWD , banks Grains, banks Boundaries (bed and bank) Grains, LWD Dominant Sediment Sources Fluvial, bank failure

  5. Application of multilinear regression analysis in modeling of soil ...

    African Journals Online (AJOL)

    The application of Multi-Linear Regression Analysis (MLRA) model for predicting soil properties in Calabar South offers a technical guide and solution in foundation designs problems in the area. Forty-five soil samples were collected from fifteen different boreholes at a different depth and 270 tests were carried out for CBR, ...

  6. application of multilinear regression analysis in modeling of soil

    African Journals Online (AJOL)

    Windows User

    Accordingly [1, 3] in their work, they applied linear regression ... (MLRA) is a statistical technique that uses several explanatory ... order to check this, they adopted bivariate correlation analysis .... groups, namely A-1 through A-7, based on their relative expected ..... Multivariate Regression in Gorgan Province North of Iran” ...

  7. Download this PDF file

    African Journals Online (AJOL)

    NWUuser

    2013-09-12

    Sep 12, 2013 ... Afr J Mar Sci 503; Gen N 434 in GG 36413 of 25 April 2013 1-2; Hauck and Sowman Waves of. Change .... definition of the term in the MLRA emphasises the element of consumptive use as ... In other words, fishing activities are ... fishers that is the primary focus of this contribution and it is therefore useful to.

  8. Vanadium NMR Chemical Shifts of (Imido)vanadium(V) Dichloride Complexes with Imidazolin-2-iminato and Imidazolidin-2-iminato Ligands: Cooperation with Quantum-Chemical Calculations and Multiple Linear Regression Analyses.

    Science.gov (United States)

    Yi, Jun; Yang, Wenhong; Sun, Wen-Hua; Nomura, Kotohiro; Hada, Masahiko

    2017-11-30

    The NMR chemical shifts of vanadium ( 51 V) in (imido)vanadium(V) dichloride complexes with imidazolin-2-iminato and imidazolidin-2-iminato ligands were calculated by the density functional theory (DFT) method with GIAO. The calculated 51 V NMR chemical shifts were analyzed by the multiple linear regression (MLR) analysis (MLRA) method with a series of calculated molecular properties. Some of calculated NMR chemical shifts were incorrect using the optimized molecular geometries of the X-ray structures. After the global minimum geometries of all of the molecules were determined, the trend of the observed chemical shifts was well reproduced by the present DFT method. The MLRA method was performed to investigate the correlation between the 51 V NMR chemical shift and the natural charge, band energy gap, and Wiberg bond index of the V═N bond. The 51 V NMR chemical shifts obtained with the present MLR model were well reproduced with a correlation coefficient of 0.97.

  9. Regional Supplement to the Corps of Engineers Wetland Delineation Manual: Western Mountains, Valleys, and Coast Region (Version 2.0)

    Science.gov (United States)

    2010-05-01

    monophylla or P. edulis), junipers (Juniperus), cottonwoods (e.g., Populus fremontii), willows (Salix), or hardwoods (e.g., Quercus , Platanus...pine in association with incense cedar and California black oak ( Quercus kelloggii) on the western slopes and Jeffrey pine on the eastern slopes...corkbark fir (Abies lasiocarpa var. arizonica ), limber pine (Pinus flexilis), and bristlecone pine (P. aristata) (Bailey 1995). Black Hills (MLRA

  10. Associated factors and prevalence of erectile dysfunction in hemodialysis patients

    Directory of Open Access Journals (Sweden)

    Marcio Rodrigues Costa

    2014-01-01

    Full Text Available Purpose: The proposal of this study was to determine the prevalence and the associated factors of erectile dysfunction (ED among hemodialysis (HD patients. Materials and Methods: This was a cross-sectional study based on data collected from HD male patients. Clinical, demographic and laboratory data of all patients were collected in three HD clinics from December 2010 to June 2011. Patients answered questions of erectile function domain from International Index of Erectile Function. Data were evaluated by descriptive analysis and by univariate (ULRA and multivariate logistic regression analysis (MLRA. Results: Three hundred and five patients participated of the study. The prevalence of ED was 68.19%. ED was associated with diabetes (DM, benign prostatic hyperplasia, glomerulonephritis as cause of chronic renal failure (CRF, smoking habits, lower creatinine levels (ULRA, use of calcium channel blocker (MLRA, aging, lower education level, alcohol consumption, DM (as cause of CRF and coronary insufficiency (ULRA and MLRA. Conclusions: ED was highly prevalent in the HD men. It was independently associated with aging, current use of alcohol, long alcohol use (even for those who do not drink more, lower education level, diabetes as cause of CRF, coronary insufficiency and use of channel blockers calcium.

  11. The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control

    Science.gov (United States)

    Lindenberg, Sandra; Klauck, Gisela; Pesavento, Christina; Klauck, Eberhard; Hengge, Regine

    2013-01-01

    C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling. PMID:23708798

  12. Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and Versant v3.0 assays for Determination of HIV-1 Viral Loads in a Cohort of Canadian Patients with Diverse HIV Subtype Infections▿

    OpenAIRE

    Church, Deirdre; Gregson, Daniel; Lloyd, Tracie; Klein, Marina; Beckthold, Brenda; Laupland, Kevin; Gill, M. John

    2010-01-01

    HIV clinics in Canada provide care to an increasing number of patients born outside of Canada with HIV-1 non-B subtype infections. Because the Easy Q HIV-1 v1.2 assay (EQ; bioMérieux) failed to detect some non-B subtype infections, a multiassay HIV-1 viral load (VL) study was conducted with patients with diverse HIV subtype infections. Patients were enrolled from the Southern Alberta HIV Clinic (SAC), Calgary, Alberta, Canada (n = 349) and the McGill HIV Clinic (MHC), Montreal, Quebec, Canada...

  13. Co-relationship between california bearing ratio and index properties of jamshoro soil

    International Nuclear Information System (INIS)

    Iqbal, F.; Kumar, A.; Murtaza, A.

    2018-01-01

    Subgrade is a most important part of a pavement structure, which should have a reasonable stiffness modulus and shear strength. CBR (California Bearing Ratio) test is performed to evaluate stiffness modulus and shear strength of subgrade soils. However, CBR test is laborious and time consuming, particularly when soil is highly plastic like Jamshoro soil. In order to overcome this limitation, it may be appropriate to correlate CBR value of soil with its index properties like grain size analysis, Atterberg limits, and compaction characteristics such as MDD (Maximum Dry Density) and OMC (Optimum Moisture Content). This paper expresses the correlations between CBR value of Jamshoro soil and its index properties. SLRA (Single Linear Regression Analysis) and MLRA(Multiple Linear Regression) based Models were utilized. It is seen that MLRA gave better correlations up to R2 of about 0.984. It is observed that the Soaked CBR value can be predicted with confidence from LL (Liquid Limit), PI (Plasticity Index) and percent finer while the un-soaked CBR value can be obtained from LL, plasticity index and MDD. (author)

  14. Bioreactor Study Employing Bacteria with Enhanced Activity toward Cyanobacterial Toxins Microcystins

    Directory of Open Access Journals (Sweden)

    Dariusz Dziga

    2014-08-01

    Full Text Available An important aim of white (grey biotechnology is bioremediation, where microbes are employed to remove unwanted chemicals. Microcystins (MCs and other cyanobacterial toxins are not industrial or agricultural pollutants; however, their occurrence as a consequence of human activity and water reservoir eutrophication is regarded as anthropogenic. Microbial degradation of microcystins is suggested as an alternative to chemical and physical methods of their elimination. This paper describes a possible technique of the practical application of the biodegradation process. The idea relies on the utilization of bacteria with a significantly enhanced MC-degradation ability (in comparison with wild strains. The cells of an Escherichia coli laboratory strain expressing microcystinase (MlrA responsible for the detoxification of MCs were immobilized in alginate beads. The degradation potency of the tested bioreactors was monitored by HPLC detection of linear microcystin LR (MC-LR as the MlrA degradation product. An open system based on a column filled with alginate-entrapped cells was shown to operate more efficiently than a closed system (alginate beads shaken in a glass container. The maximal degradation rate calculated per one liter of carrier was 219.9 µg h−1 of degraded MC-LR. A comparison of the efficiency of the described system with other biological and chemo-physical proposals suggests that this new idea presents several advantages and is worth investigating in future studies.

  15. Small-scale Fisheries Governance and Understanding the Snoek (Thyrsites atun Supply Chain in the Ocean View Fishing Community, Western Cape, South Africa

    Directory of Open Access Journals (Sweden)

    Moenieba Isaacs

    2013-12-01

    Full Text Available Postapartheid fisheries reform in South Africa, through the Marine Living Resources Act (MLRA 18 of 1998, used individual transferable quotas (ITQs to broaden resource access through allocating quotas to new entrants, even though the system has been created to reduce capacity through a reduction in the number of active fishers. The formal action space created through fisheries reform in South Africa left many artisanal fishers to operate in the informal action spaces, selling Thyrsites atun (snoek to poor communities to sustain their livelihoods. Artisanal fishers were not recognized by MLRA of 1998 and through class action case brought against the ITQ system, and in out of court settlement with the claimants in 2007, 1000 interim relief permits will be allocated to artisanal fishes and the development of a new small-scale fisheries policy for South Africa. In this case study of a fishing community in Ocean View, Cape Town I examine a snoek fishery that operates differently, through a community supply chain and informal markets, than that of the high value ITQ regulated species, yet plays a significant role in the livelihoods of artisanal fishers and in the food security of poor households. The findings of this case study show the failures of existing policy frameworks and the implications for the implementation of the new small-scale fisheries policy in South Africa.

  16. Isolation, Identification and Phenotypic Characterization of Microcystin-Degrading Bacteria from Lake Erie

    Science.gov (United States)

    Krishnan, A.; Mou, X. J.

    2015-12-01

    Lake Erie, the smallest and warmest lake among the Laurentian Great Lakes, is known for its problem of eutrophication and frequent occurrence of harmful cyanobacterial blooms (CyanoHABs). One major harmful effect of CyanoHABs is the production of cyanotoxins, especially microcystins. Microcystins (MC) are a group of hepatotoxins and the predominant variant of them is MC-LR. Field measurements and lab experiments indicate that MC degradation in Lake Erie is mainly carried out by indigenous bacteria. However, our knowledge on taxa involved in this process is very limited. This study aimed to fill this knowledge gap using a culture-dependent approach. Water and surface sediment samples were collected from Lake Erie in 2014 and 2015 and enriched with MC-LR. Cells were plated on a number of culturing media. The obtained pure bacterial cultures were screened for MC degrading abilities by MT2 BIO-LOG assays and by growing cells in liquid media containing MC-LR as the sole carbon source. In the latter experiment, MC concentrations were measured using HPLC. Isolates showing positive MC degradation activities in the screening steps were designated MC+ bacteria and characterized based on their phenotypic properties, including colony pigmentation, elevation, opacity, margin, gram nature and motility. The taxonomic identity of MC+ bacteria was determined by 16S rRNA gene full-length DNA sequencing. The presence of mlrA, a gene encoding MC cleavage pathway, was detected by PCR. Our culturing efforts obtained 520 pure cultures; 44 of them were identified as MC+. These MC+ isolates showed diversity in taxonomic identities and differed in their morphology, gram nature, colony characteristics and motility. PCR amplification of mlrA gene yield negative results for all MC+ isolates, indicating that the primers that were used may not be ubiquitous enough to cover the heterogeneity of mlrA genes or, more likely, alternative degradative genes/pathways were employed by Lake Erie bacteria

  17. Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins.

    Science.gov (United States)

    Dziga, Dariusz; Zielinska, Gabriela; Wladyka, Benedykt; Bochenska, Oliwia; Maksylewicz, Anna; Strzalka, Wojciech; Meriluoto, Jussi

    2016-03-16

    Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.

  18. Characterization of Enzymatic Activity of MlrB and MlrC Proteins Involved in Bacterial Degradation of Cyanotoxins Microcystins

    Directory of Open Access Journals (Sweden)

    Dariusz Dziga

    2016-03-01

    Full Text Available Bacterial degradation of toxic microcystins produced by cyanobacteria is a common phenomenon. However, our understanding of the mechanisms of these processes is rudimentary. In this paper several novel discoveries regarding the action of the enzymes of the mlr cluster responsible for microcystin biodegradation are presented using recombinant proteins. In particular, the predicted active sites of the recombinant MlrB and MlrC were analyzed using functional enzymes and their inactive muteins. A new degradation intermediate, a hexapeptide derived from linearized microcystins by MlrC, was discovered. Furthermore, the involvement of MlrA and MlrB in further degradation of the hexapeptides was confirmed and a corrected biochemical pathway of microcystin biodegradation has been proposed.

  19. Micro-EDM process modeling and machining approaches for minimum tool electrode wear for fabrication of biocompatible micro-components

    DEFF Research Database (Denmark)

    Puthumana, Govindan

    2017-01-01

    Micro-electrical discharge machining (micro-EDM) is a potential non-contact method for fabrication of biocompatible micro devices. This paper presents an attempt to model the tool electrode wear in micro-EDM process using multiple linear regression analysis (MLRA) and artificial neural networks...... linear regression model was developed for prediction of TWR in ten steps at a significance level of 90%. The optimum architecture of the ANN was obtained with 7 hidden layers at an R-sq value of 0.98. The predicted values of TWR using ANN matched well with the practically measured and calculated values...... (ANN). The governing micro-EDM factors chosen for this investigation were: voltage (V), current (I), pulse on time (Ton) and pulse frequency (f). The proposed predictive models generate a functional correlation between the tool electrode wear rate (TWR) and the governing micro-EDM factors. A multiple...

  20. Genetic algorithm as a variable selection procedure for the simulation of 13C nuclear magnetic resonance spectra of flavonoid derivatives using multiple linear regression.

    Science.gov (United States)

    Ghavami, Raoof; Najafi, Amir; Sajadi, Mohammad; Djannaty, Farhad

    2008-09-01

    In order to accurately simulate (13)C NMR spectra of hydroxy, polyhydroxy and methoxy substituted flavonoid a quantitative structure-property relationship (QSPR) model, relating atom-based calculated descriptors to (13)C NMR chemical shifts (ppm, TMS=0), is developed. A dataset consisting of 50 flavonoid derivatives was employed for the present analysis. A set of 417 topological, geometrical, and electronic descriptors representing various structural characteristics was calculated and separate multilinear QSPR models were developed between each carbon atom of flavonoid and the calculated descriptors. Genetic algorithm (GA) and multiple linear regression analysis (MLRA) were used to select the descriptors and to generate the correlation models. Analysis of the results revealed a correlation coefficient and root mean square error (RMSE) of 0.994 and 2.53ppm, respectively, for the prediction set.

  1. Dataset of curcumin derivatives for QSAR modeling of anti cancer against P388 cell line

    Directory of Open Access Journals (Sweden)

    Yum Eryanti

    2016-12-01

    Full Text Available The dataset of curcumin derivatives consists of 45 compounds (Table 1 with their anti cancer biological activity (IC50 against P388 cell line. 45 curcumin derivatives were used in the model development where 30 of these compounds were in the training set and the remaining 15 compounds were in the test set. The development of the QSAR model involved the use of the multiple linear regression analysis (MLRA method. Based on the method, r2 value, r2 (CV value of 0.81, 0.67 were obtained. The QSAR model was also employed to predict the biological activity of compounds in the test set. Predictive correlation coefficient r2 values of 0.88 were obtained for the test set.

  2. Statistical modeling of copper losses in the silicate slag of the sulfide concentrate smelting process

    Directory of Open Access Journals (Sweden)

    Savic Marija V.

    2015-09-01

    Full Text Available This article presents the results of the statistical modeling of copper losses in the silicate slag of the sulfide concentrates smelting process. The aim of this study was to define the correlation dependence of the degree of copper losses in the silicate slag on the following parameters of technological processes: SiO2, FeO, Fe3O4, CaO and Al2O3 content in the slag and copper content in the matte. Multiple linear regression analysis (MLRA, artificial neural networks (ANNs and adaptive network based fuzzy inference system (ANFIS were used as tools for mathematical analysis of the indicated problem. The best correlation coefficient (R2 = 0.719 of the final model was obtained using the ANFIS modeling approach.

  3. Prediction of Al2O3 leaching recovery in the Bayer process using statistical multilinear regresion analysis

    Directory of Open Access Journals (Sweden)

    Đurić I.

    2010-01-01

    Full Text Available This paper presents the results of defining the mathematical model which describes the dependence of leaching degree of Al2O3 in bauxite from the most influential input parameters in industrial conditions of conducting the leaching process in the Bayer technology of alumina production. Mathematical model is defined using the stepwise MLRA method, with R2 = 0.764 and significant statistical reliability - VIF<2 and p<0.05, on the one-year statistical sample. Validation of the acquired model was performed using the data from the following year, collected from the process conducted under industrial conditions, rendering the same statistical reliability, with R2 = 0.759.

  4. Evaluation of a Multiplex Real-Time Reverse Transcriptase PCR Assay for Detection and Differentiation of Influenza Viruses A and B during the 2001-2002 Influenza Season in Israel

    Science.gov (United States)

    Hindiyeh, Musa; Levy, Virginia; Azar, Roberto; Varsano, Noemi; Regev, Liora; Shalev, Yael; Grossman, Zehava; Mendelson, Ella

    2005-01-01

    The ability to rapidly diagnose influenza virus infections is of the utmost importance in the evaluation of patients with upper respiratory tract infections. It is also important for the influenza surveillance activities performed by national influenza centers. In the present study we modified a multiplex real-time reverse transcriptase PCR (RT-PCR) assay (which uses TaqMan chemistry) and evaluated it for its ability to detect and concomitantly differentiate influenza viruses A and B in 370 patient samples collected during the 2001-2002 influenza season in Israel. The performance of the TaqMan assay was compared to those of a multiplex one-step RT-PCR with gel detection, a shell vial immunofluorescence assay, and virus isolation in tissue culture. The TaqMan assay had an excellent sensitivity for the detection of influenza viruses compared to that of tissue culture. The overall sensitivity and specificity of the TaqMan assay compared to the results of culture were 98.4 and 85.5%, respectively. The sensitivity and specificity of the TaqMan assay for the detection of influenza virus A alone were 100 and 91.1%, respectively. On the other hand, the sensitivity and specificity for the detection of influenza virus B alone were 95.7 and 98.7%, respectively. The rapid turnaround time for the performance of the TaqMan assay (4.5 h) and the relatively low direct cost encourage the routine use of this assay in place of tissue culture. We conclude that the multiplex TaqMan assay is highly suitable for the rapid diagnosis of influenza virus infections both in well-established molecular biology laboratories and in reference clinical laboratories. PMID:15695650

  5. Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    Roberta Flávia Ribeiro Rolando

    2012-06-01

    Full Text Available This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR. A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

  6. A pilot evaluation of whole blood finger-prick sampling for point-of-care HIV viral load measurement: the UNICORN study.

    Science.gov (United States)

    Fidler, Sarah; Lewis, Heather; Meyerowitz, Jodi; Kuldanek, Kristin; Thornhill, John; Muir, David; Bonnissent, Alice; Timson, Georgina; Frater, John

    2017-10-20

    There is a global need for HIV viral load point-of-care (PoC) assays to monitor patients receiving antiretroviral therapy. UNICORN was the first study of an off-label protocol using whole blood finger-prick samples tested with and without a simple three minute spin using a clinic-room microcentrifuge. Two PoC assays were evaluated in 40 HIV-positive participants, 20 with detectable and 20 with undetectable plasma viral load (pVL) (<20 copies/ml). Using 100 µl finger-prick blood samples, the Cepheid Xpert HIV-1 Viral Load and HIV-1 Qual cartridges were compared with laboratory pVL assessment (TaqMan, Roche). For participants with undetectable viraemia by TaqMan, there was poor concordance without centrifugation with the TaqMan platform with only 40% 'undetectable' using Xpert VL and 25% 'not detected' using the Qual assay. After a 3 minute spin, 100% of samples were undetectable using either assay, showing full concordance with the TaqMan assay. Defining a lower limit of detection of 1000 copies/ml when including a spin, there was 100% concordance with the TaqMan platform with strong correlation (rho 0.95 and 0.94; p < 0.0001 for both assays). When including a simple microcentrifugation step, finger-prick PoC testing was a quick and accurate approach for assessing HIV viraemia, with excellent concordance with validated laboratory approaches.

  7. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    -point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (Delta Rn, less than or equal to 0.5). All 100 rough...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method......A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...

  8. Characterization of biofilm-forming capacity and resistance to sanitizers of a range of E. coli O26 pathotypes from clinical cases and cattle in Australia.

    Science.gov (United States)

    Lajhar, Salma A; Brownlie, Jeremy; Barlow, Robert

    2018-05-08

    The formation of biofilms and subsequent encasement of bacterial cells in a complex matrix can enhance resistance to antimicrobials and sterilizing agents making these organisms difficult to eradicate and control. The aim of this study was to evaluate and compare the capacity of 40 E. coli O26 isolates of enterohemorrhagic E. coli (EHEC, n = 27), potential EHEC (pEHEC, n = 3), atypical enteropathogenic E. coli (aEPEC, n = 8) and non-toxigenic E. coli (NTEC, n = 2) from human and cattle sources to form biofilms on different surfaces, and determine whether extracellular matrix (ECM) components (cellulose, curli), motility, prophage insertion in mlrA and cell surface hydrophobicity could influence biofilm formation. Finally, the influence of biofilm formation on the sensitivity of isolates to quaternary ammonium compounds (QACs; Profoam, Kwiksan 22) and peracetic acid-based sanitizer (Topactive Des.) for 2 min on polystyrene plate were also evaluated. Biofilm production on one surface may not indicate biofilm formation on a different surface. Biofilm was formed by different pathotypes on polystyrene (70%), stainless steel (87.5%) and glass slides (95%), however only 50% demonstrated pellicle formation. EHEC isolates were significantly more likely to form a pellicle at the air-liquid interface and biofilms on polystyrene surface at 48 h than aEPEC. Strains that don't produce ECM (curli or cellulose), harbor a prophage insertion in mlrA, and are non-motile have lower biofilm forming capacities than those isolates possessing combinations of these attributes. Hydrophobicity had no impact on biofilm formation. After 2 min exposure, none of the disinfectants tested were able to completely inactivate all cells within a biofilm regardless of pathotypes and the amount of biofilm formed. Pathotypes of E. coli O26 showed varying capacities to form biofilms, however, most EHEC strains had the capacity to form biofilm on all surfaces and at the air

  9. Characterization of mechanical behavior of a porcine pulmonary artery strip using a randomized uniaxial stretch and stretch-rate protocol

    Directory of Open Access Journals (Sweden)

    Criscione John C

    2008-01-01

    Full Text Available Abstract Background Much of the experimental work in soft tissue mechanics has been focused on fitting approximate relations for specific tissue types from aggregate data on multiple samples of the tissue. Such relations are needed for modeling applications and have reasonable predictability – especially given the natural variance in specimens. There is, however, much theoretical and experimental work to be done in determining constitutive behaviors for particular specimens and tissues. In so doing, it may be possible to exploit the natural variation in tissue ultrastructure – so to relate ultrastructure composition to tissue behavior. Thus, this study focuses on an experimental method for determining constitutive behaviors and illustrates the method with analysis of a porcine pulmonary artery strip. The method characterizes the elastic part of the response (implicitly in terms of stretch and the inelastic part in terms of short term stretch history (i.e., stretch-rate Ht2, longer term stretch history Ht1, and time since the start of testing T. Methods A uniaxial testing protocol with a random stretch and random stretch-rate was developed. The average stress at a particular stretch was chosen as the hyperelastic stress response, and deviation from the mean at this particular stretch is chosen as the inelastic deviation. Multivariable Linear Regression Analysis (MLRA was utilized to verify if Ht2, Ht1, and T are important factors for characterizing the inelastic deviation. For acquiring Ht2 and Ht1, an integral function type of stretch history was employed with time constants chosen from the relaxation spectrum of an identical size strip from the same tissue with the same orientation. Finally, statistical models that characterize the inelasticity were developed at various, nominal values of stretch, and their predictive capability was examined. Results Inelastic deviation from hyperelasticity was high (31% for low stretch and declined

  10. Characterizing the pathotype structure of barley powdery mildew and effectiveness of resistance genes to this pathogen in Kazakhstan.

    Science.gov (United States)

    Rsaliyev, Aralbek; Pahratdinova, Zhazira; Rsaliyev, Shynbolat

    2017-11-14

    Powdery mildew of barley is a wind-borne and obligate biotrophic pathogen, which ranks among the most widespread barley pathogens worldwide. However, purposeful research towards studying the structure of the barley powdery mildew populations, of their virulence and of effectiveness of certain resistance genes against the infection was not conducted in Kazakhstan till present time. This paper is the first to describe characteristics of the pathotype structure of Blumeria graminis f.sp. hordei (Bgh) population and effectiveness of resistance genes in two regions of barley cultivation in the republic. One hundred and seven isolates of Bgh were obtained from seven populations occurring on cultivated barley at two geographically locations in Kazakhstan during 2015 and 2016. Their virulence frequency was determined on 17 differential lines Pallas. All isolates were virulent on the resistance gene Mla8 and avirulent for the resistance genes Mla9, Mla1 + MlaAl2, Mla6 + Mla14, Mla13 + MlRu3, Mla7 + MlNo3, Mla10 + MlDu2, Mla13 + MlRu3 and Mlo-5. The frequencies of isolates overcoming the genes Mla3, Mla22, Mlat Mlg + MlCP and Mla12 + MlEm2 were 0.0-33.33%, and frequencies of isolates overcoming the genes Mlra, Mlk, MlLa and Mlh ranged from 10.0 to 78.6%. Based on reactions of differential lines possessing the genes Mla22, Mlra, Mlk, Mlat, MlLa and Mlh, pathotypes were identified. In total, 23 pathotypes with virulence complexity ranging from 1 to 6 were identified. During both years in all populations of South Kazakhstan and Zhambyl regions pathotypes 24 and 64 mainly prevailed. Obtained data suggest that low similarity of populations Bgh in Kazakhstan to European, African, Australian and South-East Asian populations. The present study provides a foundation for future studies on the pathogenic variability within of Bgh populations in Kazakhstan and addresses the knowledge gap on the virulence structure of Bgh in Central Asia. Complete effectiveness of the

  11. Variation in plasma calcium analysis in primary care in Sweden - a multilevel analysis

    Directory of Open Access Journals (Sweden)

    Eggertsen Robert

    2010-05-01

    Full Text Available Abstract Background Primary hyperparathyroidism (pHPT is a common disease that often remains undetected and causes severe disturbance especially in postmenopausal women. Therefore, national recommendations promoting early pHPT detection by plasma calcium (P-Ca have been issued in Sweden. In this study we aimed to investigate variation of P-Ca analysis between physicians and health care centres (HCCs in primary care in county of Skaraborg, Sweden. Methods In this cross sectional study of patients' records during 2005 we analysed records from 154 629 patients attending 457 physicians at 24 HCCs. We used multilevel logistic regression analysis (MLRA and adjusted for patient, physician and HCC characteristics. Differences were expressed as median odds ratio (MOR. Results There was a substantial variation in number of P-Ca analyses between both HCCs (MORHCC 1.65 [1.44-2.07] and physicians (MORphysician 1.95 [1.85-2.08]. The odds for a P-Ca analysis were lower for male patients (OR 0.80 [0.77-0.83] and increased with the number of diagnoses (OR 25.8 [23.5-28.5]. Sex of the physician had no influence on P-Ca test ordering (OR 0.93 [0.78-1.09]. Physicians under education ordered most P-Ca analyses (OR 1.69 [1.35-2.24] and locum least (OR 0.73 [0.57-0.94]. More of the variance was attributed to the physician level than the HCC level. Different mix of patients did not explain this variance between physicians. Theoretically, if a patient were able to change both GP and HCC, the odds of a P-Ca analysis would in median increase by 2.45. Including characteristics of the patients, physicians and HCCs in the MLRA model did not explain the variance. Conclusions The physician level was more important than the HCC level for the variation in P-Ca analysis, but further exploration of unidentified contextual factors is crucial for future monitoring of practice variation.

  12. Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells

    OpenAIRE

    Romain Barbet; Isabelle Peiffer; Antoinette Hatzfeld; Pierre Charbord; Jacques A. Hatzfeld

    2011-01-01

    We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs ...

  13. Improved real-time PCR assay for detection of the quarantine potato pathogen, Synchytrium endobioticum, in zonal centrifuge extracts from soil and in plants

    NARCIS (Netherlands)

    Gent-Pelzer, van M.P.E.; Krijger, M.C.; Bonants, P.J.M.

    2010-01-01

    Real-time PCR was used for quantitative detection of the potato pathogen, Synchytrium endobioticum, in different substrates: zonal centrifuge extracts, warts and different plant parts of potato. Specific primers and a TaqMan probe, designed from the internal transcribed spacer region of the

  14. Evaluation of 5 ' nuclease assay for detection of Actinobacillus pleuropneumoniae

    DEFF Research Database (Denmark)

    Angen, Øystein; Jensen, J.; Lavritsen, D. T.

    2001-01-01

    Sequence detection by the 5' nuclease TaqMan assay uses online detection of internal fluorogenic probes in closed PCR tubes. Primers and probe were chosen from a part of the omlA gene common to all serotypes of Actinobacillus pleuropneumoniae, which gave an amplicon of 92 bp, The test was evaluat...

  15. Effects of the EVCAM chemical validation library on differentiation using marker gene expression in lmouse embryonic stem cells

    Science.gov (United States)

    The adherent cell differentiation and cytotoxicity (ACDC) assay was used to profile the effects of the ECVAM EST validation chemical library (19 compounds) on J1 mouse embryonic stem cells (mESC). PCR-based TaqMan Low Density Arrays (TLDA) provided a high-content assessment of al...

  16. Development of a non invasion real-time PCR assay for the quantitation of chicken parvovirus in fecal swabs

    Science.gov (United States)

    The present study describes the development of a real time Taqman polymerase chain reaction (PCR) assay using a fluorescent labeled probe for the detection and quantitation of chicken parvovirus (ChPV) in feces. The primers and probes were designed based on the nucleotide sequence of the non struct...

  17. Quantitative detection of Fusarium spp. and its correlation with fumonisin content in maize from South African subsistence farmers

    NARCIS (Netherlands)

    Waalwijk, C.; Koch, S.H.; Ncube, E.; Allwood, J.; Flett, B.; Vries, de P.M.; Kema, G.H.J.

    2008-01-01

    A quantitative detection tool was developed to enable the monitoring of fumonisin-producing fungi in food and feed commodities. To this end, a quantitative PCR (TaqMan) was developed that targets a conserved region in the polyketide synthase gene fum1, which is involved in the biosynthesis of

  18. Lack of association between PKLR rs3020781 and NOS1AP rs7538490 and type 2 diabetes, overweight, obesity and related metabolic phenotypes in a Danish large-scale study: case-control studies and analyses of quantitative traits

    DEFF Research Database (Denmark)

    Andreasen, Camilla Helene; Mogensen, Mette S.; Borch-Johnsen, Knut

    2008-01-01

    of the variants in relation to overweight and obesity. METHODS: PKLR rs3020781 and NOS1AP rs7538490 were genotyped, using TaqMan allelic discrimination, in a combined study sample comprising a total of 16,801 and 16,913 individuals, respectively. The participants were ascertained from four different study groups...

  19. Clinical Value of Treponema pallidum Real-Time PCR for Diagnosis of Syphilis

    NARCIS (Netherlands)

    Heymans, R.; van der Helm, J. J.; de Vries, H. J. C.; Fennema, H. S. A.; Coutinho, R. A.; Bruisten, S. M.

    2010-01-01

    The diagnosis of syphilis can be complicated when it is based on diverse clinical manifestations, dark-field microscopy, and serology. In the present study, therefore, we examined the additional clinical value of a Treponema pallidum real-time TaqMan PCR for the detection of primary and secondary

  20. Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum

    NARCIS (Netherlands)

    Koek, A. G.; Bruisten, S. M.; Dierdorp, M.; van Dam, A. P.; Templeton, K.

    2006-01-01

    A real-time PCR assay with a Taqman probe was developed that targeted the polA gene of Treponema pallidum. The test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic. High sensitivities

  1. Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes

    Science.gov (United States)

    TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time PCR. One tube contained a primer-probe set specific for G. pallida (pale cyst nematode) multiplexed with another prim...

  2. Report for Detection of Biothreat Agents and Environmental Samples using the LLNL Virulence Array for DHS

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, Crystal [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Gardner, Shea [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McLoughlin, Kevin [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Thissen, James [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jackson, Paul [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2011-04-18

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. This report focuses on the design, testing and results of samples on the Virulence Array.

  3. Protamine 3 expressions in crossbred bull spermatozoa may not be ...

    African Journals Online (AJOL)

    The mRNA expression of PRM3 gene among two groups was evaluated by real time quantitative polymerase chain reaction (PCR) using TaqMan chemistry, where peptidylprolyl isomerase A (PPIA) was used as an internal control. Our finding revealed that expression of PRM3 was down regulated in poor quality semen ...

  4. EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

    Science.gov (United States)

    Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

  5. Analysis of two susceptibility SNPs in HLA region and evidence of ...

    Indian Academy of Sciences (India)

    Yosser Achour

    2017-11-28

    Nov 28, 2017 ... was carried out using real-time PCR methods by TaqMan allelic discrimination assay. A trend of significant ... genetic risk loci with relatively high statistical power from. GWAS was .... for the Social Sciences (SPSS) ver. 17.0 for ...

  6. Pharmacogenetics of clopidogrel: comparison between a standard and a rapid genetic testing.

    Science.gov (United States)

    Saracini, Claudia; Vestrini, Anna; Galora, Silvia; Armillis, Alessandra; Abbate, Rosanna; Giusti, Betti

    2012-06-01

    CYP2C19 variant alleles are independent predictors of clopidogrel response variability and occurrence of major adverse cardiovascular events in high-risk vascular patients on clopidogrel therapy. Increasing evidence suggests a combination of platelet function testing with CYP2C19 genetic testing may be more effective in identifying high-risk individuals for alternative antiplatelet therapeutic strategies. A crucial point in evaluating the use of these polymorphisms in clinical practice, besides test accuracy, is the cost of the genetic test and rapid availability of the results. One hundred acute coronary syndrome patients were genotyped for CYP2C19*2,*3,*4,*5, and *17 polymorphisms with two platforms: Verigene(®) and the TaqMan(®) system. Genotyping results obtained by the classical TaqMan approach and the rapid Verigene approach showed a 100% concordance for all the five polymorphisms investigated. The Verigene system had shorter turnaround time with respect to TaqMan. The cost of reagents for TaqMan genotyping was lower than that for the Verigene system, but the effective manual staff involvement and the relative cost resulted in higher cost for TaqMan than for Verigene. The Verigene system demonstrated good performance in terms of turnaround time and cost for the evaluation of the clopidogrel poor metabolizer status, giving genetic information in suitable time (206 min) for a therapeutic strategy decision.

  7. Diagnostic Assay for Rickettsia japonica

    Science.gov (United States)

    Hanaoka, Nozomu; Matsutani, Minenosuke; Kawabata, Hiroki; Yamamoto, Seigo; Fujita, Hiromi; Sakata, Akiko; Azuma, Yoshinao; Ogawa, Motohiko; Takano, Ai; Watanabe, Haruo; Kishimoto, Toshio; Shirai, Mutsunori; Kurane, Ichiro

    2009-01-01

    We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica–related strains. PMID:19961684

  8. Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae

    DEFF Research Database (Denmark)

    Rashid, Ridwan Bin; Ferdous, Jannataul; Tulsiani, Suhella

    2017-01-01

    is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24–1.32) and remarkable reproducibility (CV%: 1.08–3.7). Amplification efficiencies in the 89–100% range were observed. The assay is more economical than Taqman-based multiplex...

  9. Effect of methotrexate combined with ginger, silymarin or propolis on ...

    African Journals Online (AJOL)

    The present study was performed to evaluate the effect of three natural antioxidants on the adverse effect of methotrexate (MTX) in normal liver cells. TaqMan RT-PCR technology was used to estimate the mRNA expression levels for three genes after rats injection with a single dose of 20 mg/kg b.w MTX or the same MTX ...

  10. Detectie van Meloidogyne spp. in grondmonsters - een vergelijking van twee technieken

    NARCIS (Netherlands)

    Veenhuizen, P.T.M.; Schoemakers, N.; Vos, J.; Versteegen, F.; Landeweert, R.; Karssen, G.

    2007-01-01

    Hier volgen de samenvattingen van de bijeenkomst van 20 maart 2007 van de KNPV-werkgroep Meloidogyne. 1) Kwantitatieve multiplexdetectie van aaltjes; 2). Meloidogyne chitwoodi en M. fallax: vergelijking visuele beoordeling en Taqman-PCR aan pootaardappelen; 3) Detectie van Meloidogyne spp. in

  11. Diagnostic PCR: Comparative sensitivity of four probe chemistries

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Sommer, Helle Mølgaard

    2009-01-01

    Three probe chemistries: locked nucleic acid (LNA), minor groove binder (MGB) and Scorpion were compared with a TaqMan probe in a validated real-time PCR assay for detection of food-borne thermotolerant Campylobacter. The LNA probe produced significantly lower Ct-values and a higher proportion of...

  12. A New Diagnostic system for Ultra Sensitive and Specific Detection and Quantitation of “Candidatus Liberibacter asiaticus”, the Bacterium Associated with Citrus Huanglongbing

    Science.gov (United States)

    In this study, an ultra sensitive and quantitative diagnostic system for “Candidatus Liberibacter asiaticus” was developed. This system adapts a nested PCR and Taq-Man PCR in a single closed tube. The procedure involves two steps of PCR using the species specific outer and inner primer pairs. Differ...

  13. Comparison of Gull Feces-specific Assays Targeting the 16S rRNA Gene of Catellicoccus Marimammalium and Streptococcus spp.

    Science.gov (United States)

    Two novel gull-specific qPCR assays were developed using 16S rRNA gene sequences from gull fecal clone libraries: a SYBR-green-based assay targeting Streptococcus spp. (i.e., gull3) and a TaqMan qPCR assay targeting Catellicoccus marimammalium (i.e., gull4). The main objectives ...

  14. Determination of the species specificity of the primers for the detection of chicken and turkey meat by realtime PCR method

    Directory of Open Access Journals (Sweden)

    Lenka Maršálková

    2014-07-01

    Full Text Available The aim of this work was to use TaqMan Real-Time PCR for quantitative authentication of chicken and turkey meat. To meet this purpose, a specific pair of primers and TaqMan probe was used. The test was aimed at identifying the reaction cycle of turkey and chicken meat using by two sets of primers. With first set of primer designed for chicken we obtained the following results: Cp = 16.18 for 100% chicken DNA Cp = 29, 18 100% turkey DNA It was also amplified DNA of pig that exceeded the detection threshold fluorescence intensities in the 31.07 cycle (Cp = 31.07. Using primers designed for turkey we obtained the following results Cp = 31.16 for 100% CHDNA, Cp =16.18 100% TDNA. It was also amplified the 100% DNA of rabbit in 31.63 cycle (Cp = 31.63 and deer in cycle 32 (Cp = 32. The DNA of all other animal species was amplificated after more than 35 cycles (Cp >35. It follows that the second detection primer pair is specific enough to unrelated species of animals by 30 cycles of the reaction. Species authentication based on DNA analysis from this perspective overcomes all the shortcomings of proteins. At present, DNA analysis use different types of PCR. Is the most progressive Real-time PCR, which is suitable for the specific use of detection (primers and TaqMan probe. The TaqMan Real-time PCR is within the sensitivity and specificity, clearly one of the best methods for identifying the species of chicken and turkey meat. The specificity of this method, however, depends primarily on the specificity of the primers and TaqMan probe. The 30 cycle reaction was chosen by us as the threshold for specificity using primers for authentication chicken and turkey meat.

  15. The cobas p 630 instrument: a dedicated pre-analytic solution to optimize COBAS® AmpliPrep/COBAS® TaqMan® system workflow and turn-around-time.

    Science.gov (United States)

    Vallefuoco, L; Sorrentino, R; Spalletti Cernia, D; Colucci, G; Portella, G

    2012-12-01

    The cobas p 630, a fully automated pre-analytical instrument for primary tube handling recently introduced to complete the Cobas(®) TaqMan systems portfolio, was evaluated in conjunction with: the COBAS(®) AmpliPrep/COBAS(®) TaqMan HBV Test, v2.0, COBAS(®) AmpliPrep/COBAS(®) TaqMan HCV Test, v1.0 and COBAS(®) AmpliPrep/COBAS(®) TaqMan HIV Test, v2.0. The instrument performance in transferring samples from primary to secondary tubes, its impact in improving COBAS(®) AmpliPrep/COBAS(®) TaqMan workflow and hands-on reduction and the risk of possible cross-contamination were assessed. Samples from 42 HBsAg positive, 42 HCV and 42 HIV antibody (Ab) positive patients as well as 21 healthy blood donors were processed with or without automated primary tubes. HIV, HCV and HBsAg positive samples showed a correlation index of 0.999, 0.987 and of 0.994, respectively. To assess for cross-contamination, high titer HBV DNA positive samples, HCV RNA and HIV RNA positive samples were distributed in the cobas p 630 in alternate tube positions, adjacent to negative control samples within the same rack. None of the healthy donor samples showed any reactivity. Based on these results, the cobas p 630 can improve workflow and sample tracing in laboratories performing molecular tests, and reduce turnaround time, errors, and risks. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. The Use of Principal Component Analysis for Source Identification of PM2.5 from Selected Urban and Regional Background Sites in Poland

    Science.gov (United States)

    Błaszczak, Barbara

    2018-01-01

    The paper reports the results of the measurements of water-soluble ions and carbonaceous matter content in the fine particulate matter (PM2.5), as well as the contributions of major sources in PM2.5. Daily PM2.5 samples were collected during heating and non-heating season of the year 2013 in three different locations in Poland: Szczecin (urban background), Trzebinia (urban background) and Złoty Potok (regional background). The concentrations of PM2.5, and its related components, exhibited clear spatiotemporal variability with higher levels during the heating period. The share of the total carbon (TC) in PM2.5 exceeded 40% and was primarily determined by fluctuations in the share of OC. Sulfates (SO42-), nitrates (NO3-) and ammonium (NH4+) dominated in the ionic composition of PM2.5 and accounted together 34% (Szczecin), 30% (Trzebinia) and 18% (Złoty Potok) of PM2.5 mass. Source apportionment analysis, performed by PCA-MLRA model (Principal Component Analysis - Multilinear Regression Analysis), revealed that secondary aerosol, whose presence is related to oxidation of gaseous precursors emitted from fuel combustion and biomass burning, had the largest contribution in observed PM2.5 concentrations. In addition, the contribution of traffic sources together with road dust resuspension, was observed. The share of natural sources (sea spray, crustal dust) was generally lower.

  17. The Use of Principal Component Analysis for Source Identification of PM2.5 from Selected Urban and Regional Background Sites in Poland

    Directory of Open Access Journals (Sweden)

    Błaszczak Barbara

    2018-01-01

    Full Text Available The paper reports the results of the measurements of water-soluble ions and carbonaceous matter content in the fine particulate matter (PM2.5, as well as the contributions of major sources in PM2.5. Daily PM2.5 samples were collected during heating and non-heating season of the year 2013 in three different locations in Poland: Szczecin (urban background, Trzebinia (urban background and Złoty Potok (regional background. The concentrations of PM2.5, and its related components, exhibited clear spatiotemporal variability with higher levels during the heating period. The share of the total carbon (TC in PM2.5 exceeded 40% and was primarily determined by fluctuations in the share of OC. Sulfates (SO42-, nitrates (NO3- and ammonium (NH4+ dominated in the ionic composition of PM2.5 and accounted together ~34% (Szczecin, ~30% (Trzebinia and ~18% (Złoty Potok of PM2.5 mass. Source apportionment analysis, performed by PCA-MLRA model (Principal Component Analysis – Multilinear Regression Analysis, revealed that secondary aerosol, whose presence is related to oxidation of gaseous precursors emitted from fuel combustion and biomass burning, had the largest contribution in observed PM2.5 concentrations. In addition, the contribution of traffic sources together with road dust resuspension, was observed. The share of natural sources (sea spray, crustal dust was generally lower.

  18. PM sources in a highly industrialised area in the process of implementing PM abatement technology. Quantification and evolution.

    Science.gov (United States)

    Cruz Minguillón, María; Querol, Xavier; Alastuey, Andrés; Monfort, Eliseo; Vicente Miró, José

    2007-10-01

    Principal component analysis (PCA) coupled with a multilinear regression analysis (MLRA) was applied to PM(10) speciation data series (2002-2005) from four sampling sites in a highly industrialised area (ceramic production) in the process of implementing emission abatement technology. Five common factors with similar chemical profiles were identified at all the sites: mineral, regional background (influenced by the industrial estate located on the coast: an oil refinery and a power plant), sea spray, industrial 1 (manufacture and use of glaze components, including frit fusion) and road traffic. The contribution of the regional background differs slightly from site to site. The mineral factor, attributed to the sum of several sources (mainly the ceramic industry, but also with minor contributions from soil resuspension and African dust outbreaks) contributes between 9 and 11 microg m(-3) at all the sites. Source industrial 1 entails an increase in PM(10) levels between 4 and 5 microg m(-3) at the urban sites and 2 microg m(-3) at the suburban background site. However, after 2004, this source contributed less than 2 microg m(-3) at most sites, whereas the remaining sources did not show an upward or downward trend along the study period. This gradual decrease in the contribution of source industrial 1 coincides with the implementation of PM abatement technology in the frit fusion kilns of the area. This relationship enables us to assess the efficiency of the implementation of environmental technologies in terms of their impact on air quality.

  19. QSPR study of absorption maxima of organic dyes for dye-sensitized solar cells based on 3D descriptors

    Science.gov (United States)

    Xu, Jie; Zhang, Hui; Wang, Lei; Liang, Guijie; Wang, Luoxin; Shen, Xiaolin; Xu, Weilin

    2010-07-01

    A quantitative structure-property relationship (QSPR) study was performed for the prediction of the absorption maxima ( λmax) of organic dyes for dye-sensitized solar cells (DSSCs). The entire set of 70 dyes was divided into a training set of 53 dyes and a test set of 17 dyes according to Kennard and Stones algorithm. Three-dimensional (3D) descriptors were calculated to represent the dye molecules. A ten-descriptor model, with a squared correlation coefficient ( R2) of 0.9543 and a standard error of estimation ( s) of 14.7 nm, was produced by using the stepwise multilinear regression analysis (MLRA) on the training set. The reliability of the proposed model was further illustrated using various evaluation techniques: leave-one-out cross-validation procedure, randomization tests, and validation through the external test set. All descriptors involved in the model were derived solely from the chemical structure of the dye molecules, which makes the model very useful to estimate the λmax of dyes before they are actually synthesized.

  20. Reclamation technology development for western Arkansas coal refuse waste materials

    International Nuclear Information System (INIS)

    King, J.R.; Veith, D.L.

    1994-01-01

    Coal mining has been an important industry in the Arkansas River Valley Major Land Resource Area (MLRA) of western Arkansas for more than 100 yr., most of it with little regard for environmental concerns. Almost 3,640 ha. of land affected by surface coal mines cover the seven-county area, with less than 1,200 ha. currently in various stages of operation or reclamation. Since only the active mining sites must now be reclaimed by law, the remaining 2,440 ha. of abandoned land remains at the mercy of natural forces. Little topsoil exists on these sites and the coal wastes are generally acidic with a pH in the 4.0-5.5 range. Revegetation attempts under these conditions generally require continued maintenance and retreatment until an acceptable cover is achieved. If and when an acceptable vegetative cover is established, the cost frequently approaches $7,400/ha. ($3,000/acre). In an effort to resolve these issues and provide some direction for stabilizing coal waste lands, the US Department of Agriculture through its Soil Conservation Service Plant Materials Center at Boonville, Arkansas, received a Congressional Pass through administered by the US Bureau of Mines, to support a 5-yr. revegetation study on the coal mine spoils of western Arkansas. This paper reports the results through the spring of 1994 on that portion of the study dealing with the establishment of blackberries as a cash crop on coal mine spoils

  1. Lagrangian velocity correlations in homogeneous isotropic turbulence

    International Nuclear Information System (INIS)

    Gotoh, T.; Rogallo, R.S.; Herring, J.R.; Kraichnan, R.H.

    1993-01-01

    The Lagrangian velocity autocorrelation and the time correlations for individual wave-number bands are computed by direct numerical simulation (DNS) using the passive vector method (PVM), and the accuracy of the method is studied. It is found that the PVM is accurate when K max /k d ≥2 where K max is the maximum wave number carried in the simulation and k d is the Kolmogorov wave number. The Eulerian and Lagrangian time correlations for various wave-number bands are compared. At moderate to high wave number the Eulerian time correlation decays faster than the Lagrangian, and the effect of sweep on the former is observed. The time scale of the Eulerian correlation is found to be (kU 0 ) -1 while that of the Lagrangian is [∫ 0 k p 2 E(p)dp] -1/2 . The Lagrangian velocity autocorrelation in a frozen turbulent field is computed using the DIA, ALHDIA, and LRA theories and is compared with DNS measurements. The Markovianized Lagrangian renormalized approximation (MLRA) is compared with the DNS, and good agreement is found for one-time quantities in decaying turbulence at low Reynolds numbers and for the Lagrangian velocity autocorrelation in stationary turbulence at moderate Reynolds number. The effect of non-Gaussianity on the Lagrangian correlation predicted by the theories is also discussed

  2. Performance evaluation of cement-stabilized pond ash-rice husk ash-clay mixture as a highway construction material

    Directory of Open Access Journals (Sweden)

    Deepak Gupta

    2017-02-01

    Full Text Available This paper reports the results of an investigation carried out on clay soil stabilized with pond ash (PA, rice husk ash (RHA and cement. Modified Proctor compaction tests were performed in order to investigate the compaction behavior of clay, and California bearing ratio (CBR tests were performed to determine the strength characteristics of clay. For evaluation purpose, the specimens containing different amounts of admixtures were prepared. Clay was replaced with PA and RHA at a dosage of 30%–45% and 5%–20%, respectively. The influence of stabilizer types and dosages on mechanical properties of clay was evaluated. In order to study the surface morphology and crystallization characteristics of the soil samples, scanning electron microscopy (SEM and X-ray diffraction (XRD analyses were carried out, respectively. The results obtained indicated a decrease in the maximum dry density (MDD and a simultaneous increase in the optimum moisture content (OMC with the addition of PA and RHA. Multiple linear regression analysis (MLRA showed that the predicted values of CBR tests are in good agreement with the experimental values. Developed stabilized soil mixtures showed satisfactory strength and can be used for construction of embankments and stabilization of sub-grade soil. The use of locally available soils, PA, RHA, and cement in the production of stabilized soils for such applications can provide sustainability for the local construction industry.

  3. Rangeland Brush Estimation Toolbox (RaBET): An Approach for Evaluating Brush Management Conservation Efforts in Western Grazing Lands

    Science.gov (United States)

    Holifield Collins, C.; Kautz, M. A.; Skirvin, S. M.; Metz, L. J.

    2016-12-01

    There are over 180 million hectares of rangelands and grazed forests in the central and western United States. Due to the loss of perennial grasses and subsequent increased runoff and erosion that can degrade the system, woody cover species cannot be allowed to proliferate unchecked. The USDA-Natural Resources Conservation Service (NRCS) has allocated extensive resources to employ brush management (removal) as a conservation practice to control woody species encroachment. The Rangeland-Conservation Effects Assessment Project (CEAP) has been tasked with determining how effective the practice has been, however their land managers lack a cost-effective means to conduct these assessments at the necessary scale. An ArcGIS toolbox for generating large-scale, Landsat-based, spatial maps of woody cover on grazing lands in the western United States was developed through a collaboration with NRCS Rangeland-CEAP. The toolbox contains two main components of operation, image generation and temporal analysis, and utilizes simple interfaces requiring minimum user inputs. The image generation tool utilizes geographically specific algorithms developed from combining moderate-resolution (30-m) Landsat imagery and high-resolution (1-m) National Agricultural Imagery Program (NAIP) aerial photography to produce the woody cover scenes at the Major Land Resource (MLRA) scale. The temporal analysis tool can be used on these scenes to assess treatment effectiveness and monitor woody cover reemergence. RaBET provides rangeland managers an operational, inexpensive decision support tool to aid in the application of brush removal treatments and assessing their effectiveness.

  4. Design and statistical optimization of glipizide loaded lipospheres using response surface methodology.

    Science.gov (United States)

    Shivakumar, Hagalavadi Nanjappa; Patel, Pragnesh Bharat; Desai, Bapusaheb Gangadhar; Ashok, Purnima; Arulmozhi, Sinnathambi

    2007-09-01

    A 32 factorial design was employed to produce glipizide lipospheres by the emulsification phase separation technique using paraffin wax and stearic acid as retardants. The effect of critical formulation variables, namely levels of paraffin wax (X1) and proportion of stearic acid in the wax (X2) on geometric mean diameter (dg), percent encapsulation efficiency (% EE), release at the end of 12 h (rel12) and time taken for 50% of drug release (t50), were evaluated using the F-test. Mathematical models containing only the significant terms were generated for each response parameter using the multiple linear regression analysis (MLRA) and analysis of variance (ANOVA). Both formulation variables studied exerted a significant influence (p optimization using the desirability approach was employed to develop an optimized formulation by setting constraints on the dependent and independent variables. The experimental values of dg, % EE, rel12 and t50 values for the optimized formulation were found to be 57.54 +/- 1.38 mum, 86.28 +/- 1.32%, 77.23 +/- 2.78% and 5.60 +/- 0.32 h, respectively, which were in close agreement with those predicted by the mathematical models. The drug release from lipospheres followed first-order kinetics and was characterized by the Higuchi diffusion model. The optimized liposphere formulation developed was found to produce sustained anti-diabetic activity following oral administration in rats.

  5. Endosperm imprinting: a child custody battle?

    Science.gov (United States)

    Becraft, Philip W

    2012-02-07

    Endosperm gene imprinting has long been speculated to control nutrient allocation to seeds. For the first time, an imprinted gene directly involved in this process has been identified. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Interim Report on SNP analysis and forensic microarray probe design for South American hemorrhagic fever viruses, tick-borne encephalitis virus, henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever viruses, Rift Valley fever

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C; Gardner, S

    2012-06-05

    The goal of this project is to develop forensic genotyping assays for select agent viruses, enhancing the current capabilities for the viral bioforensics and law enforcement community. We used a multipronged approach combining bioinformatics analysis, PCR-enriched samples, microarrays and TaqMan assays to develop high resolution and cost effective genotyping methods for strain level forensic discrimination of viruses. We have leveraged substantial experience and efficiency gained through year 1 on software development, SNP discovery, TaqMan signature design and phylogenetic signature mapping to scale up the development of forensics signatures in year 2. In this report, we have summarized the whole genome wide SNP analysis and microarray probe design for forensics characterization of South American hemorrhagic fever viruses, tick-borne encephalitis viruses and henipaviruses, Old World Arenaviruses, filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus and Japanese encephalitis virus.

  7. Report for the NGFA-5 project.

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, C; Jackson, P; Thissen, J; Wollard, J; Gardner, S; McLoughlin, K

    2011-11-15

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, TaqMan PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. To effectively compare the sensitivity and specificity of the different genomic technologies, we used SNP TaqMan PCR, MLVA, microarray and high-throughput illumine and 454 sequencing to test various strains from B. anthracis, B. thuringiensis, BioWatch aerosol filter extracts or soil samples that were spiked with B. anthracis, and samples that were previously collected during DHS and EPA environmental release exercises that were known to contain B. thuringiensis spores. The results of all the samples against the various assays are discussed in this report.

  8. Longitudinal Quantitative Detection by Real-Time PCR of Mycoplasma genitalium in First-Pass Urine of Men with Recurrent Nongonococcal Urethritis

    Science.gov (United States)

    Deguchi, Takashi; Yoshida, Takashi; Yokoi, Shigeaki; Ito, Masayasu; Tamaki, Masayoshi; Ishiko, Hiroaki; Maeda, Shin-Ichi

    2002-01-01

    By using a TaqMan assay we monitored longitudinal changes in Mycoplasma genitalium loads in five men with recurrent M. genitalium-positive nongonococcal urethritis. We observed regrowth of M. genitalium persisting in hosts after treatment and a possible association of the increase in the M. genitalium load with emergence of symptoms and signs of nongonococcal urethritis in four of these patients. PMID:12354899

  9. Allele Frequency - JSNP | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available nd 39 SNPs are assayed in three (POP_*) and two (RIKEN_japanese_*) panels, respectively. Derived from Flat f... assay (JBIC-allele and RIKEN_japanese_*), TaqMan assay (RIKEN-allele) or direct sequencing / allelic discri...unteers under informed consent RIKEN_japanese_normal_weight - 711 unrelated japanese normal weight volunteer...s ( body mass index RIKEN_japanese_obese - 796 unrelated japanese obese patients

  10. Development of Medical Technology for Contingency Response to Marrow Toxic Agents

    Science.gov (United States)

    2016-10-07

    Purified genomic DNA absorbed onto cotton- tipped swabs (“DNA swabs”) has proven to be successful and cost-effective alternative QC sample type...inversely correlated with the ability to achieve long range PCR amplification of the DNA obtained from the cotton tipped swab used by NMDP for donor...Harvey, C. Steward and V. Rocha 800 pairs Microsatellit es and STR Taqman In process Prognostic impact of somatic mutation and the levels of

  11. Development of Medical Technology for Contingency Response to Marrow Toxic Agents

    Science.gov (United States)

    2018-01-18

    Antigen will Result in Robust Serologic Response to H-Y Antigens in Male HSC transplantation Recipients D. Miklos 288 pairs H-Y Antigen ELISA ...Arai, D. Miklos 200 pairs Anti-body ELISA , Protein array Yes Detection of Donor- Directed, HLA-Specific Alloantibodies in Recipients of...SNPs ELISA and Taqman Yes Natural killer cell genomics and outcomes after allogeneic transplantation for lymphoma V. Bachanova, J. Miller

  12. Molecular diagnosis of lyssaviruses and sequence comparison of Australian bat lyssavirus samples.

    Science.gov (United States)

    Foord, A J; Heine, H G; Pritchard, L I; Lunt, R A; Newberry, K M; Rootes, C L; Boyle, D B

    2006-07-01

    To evaluate and implement molecular diagnostic tests for the detection of lyssaviruses in Australia. A published hemi-nested reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of all lyssavirus genotypes was modified to a fully nested RT-PCR format and compared with the original assay. TaqMan assays for the detection of Australian bat lyssavirus (ABLV) were compared with both the nested and hemi-nested RT-PCR assays. The sequences of RT-PCR products were determined to assess sequence variations of the target region (nucleocapsid gene) in samples of ABLV originating from different regions. The nested RT-PCR assay was highly analytically specific, and at least as analytically sensitive as the hemi-nested assay. The TaqMan assays were highly analytically specific and more analytically sensitive than either RT-PCR assay, with a detection level of approximately 10 genome equivalents per microl. Sequence of the first 544 nucleotides of the nucleocapsid protein coding sequence was obtained from all samples of ABLV received at Australian Animal Health Laboratory during the study period. The nested RT-PCR provided a means for molecular diagnosis of all tested genotypes of lyssavirus including classical rabies virus and Australian bat lyssavirus. The published TaqMan assay proved to be superior to the RT-PCR assays for the detection of ABLV in terms of analytical sensitivity. The TaqMan assay would also be faster and cross contamination is less likely. Nucleotide sequence analyses of samples of ABLV from a wide geographical range in Australia demonstrated the conserved nature of this region of the genome and therefore the suitability of this region for molecular diagnosis.

  13. Molecular diagnostics for the detection of Bokeloh bat lyssavirus in a bat from Bavaria, Germany.

    Science.gov (United States)

    Freuling, Conrad M; Abendroth, Björn; Beer, Martin; Fischer, Melina; Hanke, Dennis; Hoffmann, Bernd; Höper, Dirk; Just, Frank; Mettenleiter, Thomas C; Schatz, Juliane; Müller, Thomas

    2013-11-06

    A brain sample of a Natterer's bat tested positive for rabies with classical virological techniques. Molecular techniques confirmed the presence of Bokeloh bat lyssavirus (BBLV) in Germany for the second time. Sequence analysis revealed a close genetic relationship to the initial German BBLV case. Using a TaqMan RT-PCR specific for BBLV viral RNA was detected in various other organs albeit with differences in the relative viral load. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Relationship Between Genotype Variants Follicle-stimulating Hormone Receptor Gene Polymorphisms (FSHR) and Morphology of Oocytes Prior to ICSI Procedures

    OpenAIRE

    Gashi, Zafer; Elezaj, Shkelzen; Zeqiraj, Afrim; Grabanica, Driton; Shabani, Isak; Gruda, Bujar; Gashi, Fitore

    2016-01-01

    Introduction: This study investigated association of Asn680Ser FSHR polymorphism with the ovarian response in 104 women of Albanian ethnic population enrolled in ICSI program. The reason of infertility in all cases has been identified as male factor. Methods: Analysis of the Asn680Ser polymorphism was performed using TaqMan? SNP Genotyping Assay. Clinical and endocrinologic parameters were analyzed based on the genotype, age, BMI, oocyte yield, number of transferred embryos and pregnancy rate...

  15. Monitoring and improving the sensitivity of dengue nested RT-PCR used in longitudinal surveillance in Thailand.

    Science.gov (United States)

    Klungthong, Chonticha; Manasatienkij, Wudtichai; Phonpakobsin, Thipwipha; Chinnawirotpisan, Piyawan; Rodpradit, Prinyada; Hussem, Kittinun; Thaisomboonsuk, Butsaya; Ong-ajchaowlerd, Prapapun; Nisalak, Ananda; Kalayanarooj, Siripen; Buddhari, Darunee; Gibbons, Robert V; Jarman, Richard G; Yoon, In-Kyu; Fernandez, Stefan

    2015-02-01

    AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013. The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods. Published by Elsevier B.V.

  16. Investigation of Ureaplasma urealyticum biovars and their relationship with antimicrobial resistance

    Directory of Open Access Journals (Sweden)

    Zhu Chang-tai

    2011-01-01

    Full Text Available Purpose: To develop Taqman fluorescence quantitative polymerase chain reaction (PCR method for investigating the characteristics of the distributions of Ureaplasma urealyticum (UU biovars and to explore the relationship between UU biovars and antimicrobial resistance. Materials and Methods: By the method of culture, Ureaplasma species were detected. Taqman fluorescence quantitative PCR for detecting UU biovars were developed and UU clinical isolates were detected to distinguish biovars. The broth micro-dilution susceptibility testing methods were used to determine UU susceptibility. Results: By Taqman PCR method, UU biovars was successfully detected. Of 126 samples, biovar 1 was found in 73 (57.94%. There was a statistical difference between genital-urinary tract infection group and asymptomatic group (P<0.05. In the region, UU biovar 1 to 9 kinds of agents kept higher susceptibility rates, but biovar 2 maintained higher susceptibility rates only to tetracyclines. Compared with biovar 1, UU biovar 2 resistance rates to 7 kinds of agents were higher (P<0.05. Conclusions: (1 Our new established Taqman PCR method is a useful tool for screening UU biovars. (2 UU biovar 1 predominated in asymptomatic population; whereas in genital-urinary tract infection population UU biovar 2 had a higher proportion. (3 The characteristics of drug resistance were different between UU biovars. Overall, both two biovars remained higher susceptibility rates to tetracyclines. A majority of biovor 1 strains were sensitive to macrolides and quinolones; while only a small number of biovar 2 strains kept sensitive to roxithromycin and quinolones, a large proportion of biovar 2 strains were found in intermediate ranges.

  17. Real-time PCR for type-specific identification of herpes simplex in clinical samples: evaluation of type-specific results in the context of CNS diseases.

    Science.gov (United States)

    Meylan, Sylvain; Robert, Daniel; Estrade, Christine; Grimbuehler, Valérie; Péter, Olivier; Meylan, Pascal R; Sahli, Roland

    2008-02-01

    HSV-1 and HSV-2 cause CNS infections of dissimilar clinico-pathological characteristics with prognostic and therapeutic implications. To validate a type-specific real-time PCR that uses MGB/LNA Taqman probes and to review the virologico-clinical data of 25 eligible patients with non-neonatal CNS infections. This real-time PCR was evaluated against conventional PCR (26 CSF and 20 quality controls), and LightCycler assay (51 mucocutaneous, 8 CSF and 32 quality controls) and culture/immunofluorescence (75 mucocutaneous) to assess typing with independent methods. Taqman real-time PCR detected 240 HSV genomes per ml CSF, a level appropriate for the management of patients, and provided unambiguous typing for the 104 positive (62 HSV-1 and 42 HSV-2) out the 160 independent clinical samples tested. HSV type diagnosed by Taqman real-time PCR predicted final diagnosis (meningitis versus encephalitis/meningoencephalitis, p<0.001) in 24/25 patients at time of presentation, in contrast to clinical evaluation. Our real-time PCR, as a sensitive and specific means for type-specific HSV diagnosis, provided rapid prognostic information for patient management.

  18. Evaluation of CYP1A1 and CYP2B1/2 m-RNA induction in rat liver slices using the NanoString technology: a novel tool for drug discovery lead optimization.

    Science.gov (United States)

    Palamanda, Jairam R; Kumari, Pramila; Murgolo, Nicholas; Benbow, Larry; Lin, Xinjie; Nomeir, Amin A

    2009-08-01

    Cytochrome P450 (CYP) induction in rodents and humans is considered a liability for new chemical entities (NCEs) in drug discovery. In particular, CYP1A1 and CYP2B1/2 have been associated with the induction of liver tumors in oncogenicity studies during safety evaluation studies of potential drugs. In our laboratory, real time PCR (Taqman) has been used to quantify the induction of rat hepatic CYP1A1 and CYP2B1/2 in precision -cut rat liver slices. A novel technology that does not require m-RNA isolation or RT-PCR, (developed by NanoString Technologies) has been investigated to quantify CYP1A1 and CYP2B1/2 induction in rat liver slices. Seventeen commercially available compounds were evaluated using both Taqman and NanoString technologies. Precision-cut rat liver slices were incubated with individual compounds for 24 hr at 37 degrees C in a humidified CO(2) incubator and CYP1A1 and CYP2B1/2 m-RNA were quantified. The results from the NanoString technology were similar to those of the Taqman(R) with a high degree of correlation for both CYP isoforms (r(2)>0.85). Therefore, NanoString provides an additional new technology to evaluate the induction of CYP1A1 and 2B1/2, as well as potentially other enzymes or transporters in rat liver slices.

  19. Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

    Science.gov (United States)

    Cassler, M; Peterson, C L; Ledger, A; Pomponi, S A; Wright, A E; Winegar, R; McCarthy, P J; Lopez, J V

    2008-04-01

    In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

  20. Development of Real-Time PCR Methods for the Detection of Bacterial Meningitis Pathogens without DNA Extraction.

    Directory of Open Access Journals (Sweden)

    Jeni Vuong

    Full Text Available Neisseria meningitidis (Nm, Haemophilus influenzae (Hi, and Streptococcus pneumoniae (Sp are the lead causes of bacterial meningitis. Detection of these pathogens from clinical specimens using traditional real-time PCR (rt-PCR requires DNA extraction to remove the PCR inhibitors prior to testing, which is time consuming and labor intensive. In this study, five species-specific (Nm-sodC and -ctrA, Hi-hpd#1 and -hpd#3 and Sp-lytA and six serogroup-specific rt-PCR tests (A, B, C, W, X, Y targeting Nm capsular genes were evaluated in the two direct rt-PCR methods using PerfeCTa and 5x Omni that do not require DNA extraction. The sensitivity and specify of the two direct rt-PCR methods were compared to TaqMan traditional rt-PCR, the current standard rt-PCR method for the detection of meningitis pathogens. The LLD for all 11 rt-PCR tests ranged from 6,227 to 272,229 CFU/ml for TaqMan, 1,824-135,982 for 5x Omni, and 168-6,836 CFU/ml for PerfeCTa. The diagnostic sensitivity using TaqMan ranged from 89.2%-99.6%, except for NmB-csb, which was 69.7%. For 5x Omni, the sensitivity varied from 67.1% to 99.8%, with three tests below 90%. The sensitivity of these tests using PerfeCTa varied from 89.4% to 99.8%. The specificity ranges of the 11 tests were 98.0-99.9%, 97.5-99.9%, and 92.9-99.9% for TaqMan, 5x Omni, and PerfeCTa, respectively. PerfeCTa direct rt-PCR demonstrated similar or better sensitivity compared to 5x Omni direct rt-PCR or TaqMan traditional rt-PCR. Since the direct rt-PCR method does not require DNA extraction, it reduces the time and cost for processing CSF specimens, increases testing throughput, decreases the risk of cross-contamination, and conserves precious CSF. The direct rt-PCR method will be beneficial to laboratories with high testing volume.

  1. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Science.gov (United States)

    Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

    2007-01-01

    Background Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot

  2. Comparison of 3 estimation methods of mycophenolic acid AUC based on a limited sampling strategy in renal transplant patients.

    Science.gov (United States)

    Hulin, Anne; Blanchet, Benoît; Audard, Vincent; Barau, Caroline; Furlan, Valérie; Durrbach, Antoine; Taïeb, Fabrice; Lang, Philippe; Grimbert, Philippe; Tod, Michel

    2009-04-01

    A significant relationship between mycophenolic acid (MPA) area under the plasma concentration-time curve (AUC) and the risk for rejection has been reported. Based on 3 concentration measurements, 3 approaches have been proposed for the estimation of MPA AUC, involving either a multilinear regression approach model (MLRA) or a Bayesian estimation using either gamma absorption or zero-order absorption population models. The aim of the study was to compare the 3 approaches for the estimation of MPA AUC in 150 renal transplant patients treated with mycophenolate mofetil and tacrolimus. The population parameters were determined in 77 patients (learning study). The AUC estimation methods were compared in the learning population and in 73 patients from another center (validation study). In the latter study, the reference AUCs were estimated by the trapezoidal rule on 8 measurements. MPA concentrations were measured by liquid chromatography. The gamma absorption model gave the best fit. In the learning study, the AUCs estimated by both Bayesian methods were very similar, whereas the multilinear approach was highly correlated but yielded estimates about 20% lower than Bayesian methods. This resulted in dosing recommendations differing by 250 mg/12 h or more in 27% of cases. In the validation study, AUC estimates based on the Bayesian method with gamma absorption model and multilinear regression approach model were, respectively, 12% higher and 7% lower than the reference values. To conclude, the bicompartmental model with gamma absorption rate gave the best fit. The 3 AUC estimation methods are highly correlated but not concordant. For a given patient, the same estimation method should always be used.

  3. Validation of Quantitative Structure-Activity Relationship (QSAR Model for Photosensitizer Activity Prediction

    Directory of Open Access Journals (Sweden)

    Sharifuddin M. Zain

    2011-11-01

    Full Text Available Photodynamic therapy is a relatively new treatment method for cancer which utilizes a combination of oxygen, a photosensitizer and light to generate reactive singlet oxygen that eradicates tumors via direct cell-killing, vasculature damage and engagement of the immune system. Most of photosensitizers that are in clinical and pre-clinical assessments, or those that are already approved for clinical use, are mainly based on cyclic tetrapyrroles. In an attempt to discover new effective photosensitizers, we report the use of the quantitative structure-activity relationship (QSAR method to develop a model that could correlate the structural features of cyclic tetrapyrrole-based compounds with their photodynamic therapy (PDT activity. In this study, a set of 36 porphyrin derivatives was used in the model development where 24 of these compounds were in the training set and the remaining 12 compounds were in the test set. The development of the QSAR model involved the use of the multiple linear regression analysis (MLRA method. Based on the method, r2 value, r2 (CV value and r2 prediction value of 0.87, 0.71 and 0.70 were obtained. The QSAR model was also employed to predict the experimental compounds in an external test set. This external test set comprises 20 porphyrin-based compounds with experimental IC50 values ranging from 0.39 µM to 7.04 µM. Thus the model showed good correlative and predictive ability, with a predictive correlation coefficient (r2 prediction for external test set of 0.52. The developed QSAR model was used to discover some compounds as new lead photosensitizers from this external test set.

  4. Development of chimeric gene promoters responsive to hypoxia and ionizing radiation

    International Nuclear Information System (INIS)

    Zheng Aiqing; Yu Jinming

    2004-01-01

    The authors describe two systems that make use of gene-directed enzyme prodrug therapy, regulated by radiation or hypoxic-responsive promoters. The use of treatment-, condition- or tumor-specific promoters to control gene-directed enzyme prodrug therapy is one such method for targeting gene expression to the tumor. The development of such strategies that achieve tumor targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy

  5. Potential role of miR-9 and miR-223 in recurrent ovarian cancer

    Directory of Open Access Journals (Sweden)

    McGuinness Eamonn

    2008-04-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are small, noncoding RNAs that negatively regulate gene expression by binding to target mRNAs. miRNAs have not been comprehensively studied in recurrent ovarian cancer, yet an incurable disease. Results Using real-time RT-PCR, we obtained distinct miRNA expression profiles between primary and recurrent serous papillary ovarian adenocarcinomas (n = 6 in a subset of samples previously used in a transcriptome approach. Expression levels of top dysregulated miRNA genes, miR-223 and miR-9, were examined using TaqMan PCR in independent cohorts of fresh frozen (n = 18 and FFPE serous ovarian tumours (n = 22. Concordance was observed on TaqMan analysis for miR-223 and miR-9 between the training cohort and the independent test cohorts. Target prediction analysis for the above miRNA "recurrent metastatic signature" identified genes previously validated in our transcriptome study. Common biological pathways well characterised in ovarian cancer were shared by miR-9 and miR-223 lists of predicted target genes. We provide strong evidence that miR-9 acts as a putative tumour suppressor gene in recurrent ovarian cancer. Components of the miRNA processing machinery, such as Dicer and Drosha are not responsible for miRNA deregulation in recurrent ovarian cancer, as deluded by TaqMan and immunohistochemistry. Conclusion We propose a miRNA model for the molecular pathogenesis of recurrent ovarian cancer. Some of the differentially deregulated miRNAs identified correlate with our previous transcriptome findings. Based on integrated transcriptome and miRNA analysis, miR-9 and miR-223 can be of potential importance as biomarkers in recurrent ovarian cancer.

  6. Development and utility of an internal threshold control (ITC real-time PCR assay for exogenous DNA detection.

    Directory of Open Access Journals (Sweden)

    Weiyi Ni

    Full Text Available Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct or negative (i.e. Ct greater than the ITC Ct. The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.

  7. Identification of G-Protein-Coupled Receptors (GPCRs) in Pulmonary Artery Smooth Muscle Cells as Novel Therapeutic Targets

    Science.gov (United States)

    2017-10-01

    who do not have pulmonary hypertension (PH) Completed 12/2015 lb. Prepare RNA and cDNA from PASMCs and use Taqman GPCR arrays to identify and quantify...expression of GPCRs with known physiologic agonists .Completed 5/2017 le. Prepare RNA and cDNA from commercially available coronary artery and...Isolate and culture PASMCs from 8 (4 male; 4 female) 3 -month old rats and mice, prepare RNA and cDNA and assess expression of GPCRs with known

  8. Association of DPP4 Gene Polymorphisms with Type 2 Diabetes Mellitus in Malaysian Subjects

    OpenAIRE

    Ahmed, Radwan H.; Huri, Hasniza Zaman; Al-Hamodi, Zaid; Salem, Sameer D.; Al-absi, Boshra; Muniandy, Sekaran

    2016-01-01

    Background Genetic polymorphisms of the Dipeptidyl Peptidase 4 (DPP4) gene may play a role in the etiology of type 2 diabetes mellitus (T2DM). This study aimed to investigate the possible association of single nucleotide polymorphisms (SNPs) of the DPP4 gene in Malaysian subjects with T2DM and evaluated whether they had an effect on the serum levels of soluble dipeptidyl peptidase 4 (sDPP-IV). Method Ten DPP4 SNPs were genotyped by TaqMan genotyping assays in 314 subjects with T2DM and 235 co...

  9. Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture.

    Science.gov (United States)

    de Wit, C; Fautz, C; Xu, Y

    2000-09-01

    Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1x10(6) particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3-6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5'-->3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600 particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell

  10. Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India

    Directory of Open Access Journals (Sweden)

    Aman Kamboj

    2014-01-01

    Full Text Available Crimean-Congo hemorrhagic fever (CCHF is an emerging zoonotic disease in India and requires immediate detection of infection both for preventing further transmission and for controlling the infection. The present study describes development, optimization, and evaluation of a novel molecular beacon-based real-time RT-PCR assay for rapid, sensitive, and specific diagnosis of Crimean-Congo hemorrhagic fever virus (CCHFV. The developed assay was found to be a better alternative to the reported TaqMan assay for routine diagnosis of CCHF.

  11. Molecular Detection of Leptospiral DNA in Environmental Water on St. Kitts

    Directory of Open Access Journals (Sweden)

    Julienne Rawlins

    2014-08-01

    Full Text Available Leptospirosis is an important waterborne zoonotic disease caused by pathogenic Leptospira. The pathogen is maintained in a population due to chronic colonization and shedding from renal tubules of domestic and wild animals. Humans and other animals become infected when they come in contact with urine from infected animals, either directly or through urine-contaminated surface water. In this study, we screened environmental water on the island of St. Kitts by using a TaqMan based real time quantitative polymerase chain reaction (qPCR targeting a pathogen specific leptospiral gene, lipl32. Our results indicate that around one-fifth of tested water sources have detectable leptospiral DNA.

  12. Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform.

    Science.gov (United States)

    Opota, Onya; Brouillet, René; Greub, Gilbert; Jaton, Katia

    2017-01-01

    The advances in molecular biology of the last decades have dramatically improved the field of diagnostic bacteriology. In particular, PCR-based technologies have impacted the diagnosis of infections caused by obligate intracellular bacteria such as pathogens from the Chlamydiacae family. Here, we describe a real-time PCR-based method using the Taqman technology for the diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus infection. The method presented here can be applied to various clinical samples and can be adapted on opened molecular diagnostic platforms.

  13. Real-time PCR genotyping assay for canine progressive rod-cone degeneration and mutant allele frequency in Toy Poodles, Chihuahuas and Miniature Dachshunds in Japan

    OpenAIRE

    KOHYAMA, Moeko; TADA, Naomi; MITSUI, Hiroko; TOMIOKA, Hitomi; TSUTSUI, Toshihiko; YABUKI, Akira; RAHMAN, Mohammad Mahbubur; KUSHIDA, Kazuya; MIZUKAMI, Keijiro; YAMATO, Osamu

    2015-01-01

    Canine progressive rod-cone degeneration (PRCD) is a middle- to late-onset, autosomal recessive, inherited retinal disorder caused by a substitution (c.5G>A) in the canine PRCD gene that has been identified in 29 or more purebred dogs. In the present study, a TaqMan probe-based real-time PCR assay was developed and evaluated for rapid genotyping and large-scale screening of the mutation. Furthermore, a genotyping survey was carried out in a population of the three most popular breeds in Japan...

  14. Apolipoprotein(a) Kringle-IV Type 2 Copy Number Variation Is Associated with Venous Thromboembolism

    DEFF Research Database (Denmark)

    Sticchi, Elena; Magi, Alberto; Kamstrup, Pia R

    2016-01-01

    (a), including the kringle-IV type 2 (KIV-2) size polymorphism. Aim of the study was to investigate the role of LPA gene KIV-2 size polymorphism and single nucleotide polymorphisms (SNPs) (rs1853021, rs1800769, rs3798220, rs10455872) in modulating VTE susceptibility. Five hundred and sixteen patients with VTE...... without hereditary and acquired thrombophilia and 1117 healthy control subjects, comparable for age and sex, were investigated. LPA KIV-2 polymorphism, rs3798220 and rs10455872 SNPs were genotyped by TaqMan technology. Concerning rs1853021 and rs1800769 SNPs, PCR-RFLP assay was used. LPA KIV-2 repeat...

  15. AHSG tag single nucleotide polymorphisms associate with type 2 diabetes and dyslipidemia: studies of metabolic traits in 7,683 white Danish subjects

    DEFF Research Database (Denmark)

    Andersen, Gitte; Burgdorf, Kristoffer Sølvsten; Sparsø, Thomas

    2008-01-01

    been largely successful. We related seven frequent AHSG tag single nucleotide polymorphisms to a range of metabolic traits, including type 2 diabetes, obesity, and dyslipidemia. RESEARCH DESIGN AND METHODS: The polymorphisms were genotyped in 7,683 white Danish subjects using Taqman allelic...... with dyslipidemia (P = 0.003 and P(corr) = 0.009). Thr248Met (rs4917) tended to associate with lower fasting and post-oral glucose tolerance test serum insulin release (P = 0.02, P(corr) = 0.1 for fasting and P = 0.04, P(corr) = 0.2 for area under the insulin curve) and improved insulin sensitivity estimated...

  16. PKD2 mutation in an Iranian autosomal dominant polycystic kidney disease family with misleading linkage analysis data

    Directory of Open Access Journals (Sweden)

    Mona Entezam

    2016-06-01

    Conclusion: Although analysis of additive informative polymorphic markers can overcome the misleading haplotype data, it is limited because of the lack of other highly polymorphic microsatellite markers closer to the gene. Direct mutation screening can identify the causative mutation in the apparently unlinked pedigree; moreover, it is the only approach to achieve the confirmed diagnosis in individuals with equivocal imaging results.

  17. Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [ChIP-Seq

    NARCIS (Netherlands)

    Muiño, J.M.; Bruijn, de S.A.; Vingron, Martin; Angenent, G.C.; Kaufmann, K.

    2015-01-01

    Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the

  18. Distribution and evolution of genes responsible for biosynthesis of mycotoxins in Fusarium

    Science.gov (United States)

    Fusarium secondary metabolites (SMs) include some of the mycotoxins of greatest concern to food and feed safety. In fungi, genes directly involved in synthesis of the same SM are typically located adjacent to one another in gene clusters. To better understand the distribution and evolution of mycoto...

  19. Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

    NARCIS (Netherlands)

    Krens, F.A.; Schaart, J.G.; Groenwold, R.; Walraven, A.E.J.; Hesselink, T.; Thissen, J.T.N.M.

    2011-01-01

    Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars.

  20. Application of a 5-tiered scheme for standardized classification of 2,360 unique mismatch repair gene variants in the InSiGHT locus-specific database

    NARCIS (Netherlands)

    Thompson, Bryony A.; Spurdle, Amanda B.; Plazzer, John-Paul; Greenblatt, Marc S.; Akagi, Kiwamu; Al-Mulla, Fahd; Bapat, Bharati; Bernstein, Inge; Capella, Gabriel; den Dunnen, Johan T.; du Sart, Desiree; Fabre, Aurelie; Farrell, Michael P.; Farrington, Susan M.; Frayling, Ian M.; Frebourg, Thierry; Goldgar, David E.; Heinen, Christopher D.; Holinski-Feder, Elke; Kohonen-Corish, Maija; Robinson, Kristina Lagerstedt; Leung, Suet Yi; Martins, Alexandra; Moller, Pal; Morak, Monika; Nystrom, Minna; Peltomaki, Paivi; Pineda, Marta; Qi, Ming; Ramesar, Rajkumar; Rasmussen, Lene Juel; Royer-Pokora, Brigitte; Scott, Rodney J.; Sijmons, Rolf; Tavtigian, Sean V.; Tops, Carli M.; Weber, Thomas; Wijnen, Juul; Woods, Michael O.; Macrae, Finlay; Genuardi, Maurizio

    The clinical classification of hereditary sequence variants identified in disease-related genes directly affects clinical management of patients and their relatives. The International Society for Gastrointestinal Hereditary Tumours (InSiGHT) undertook a collaborative effort to develop, test and

  1. Evolutionary dynamics of DNA-binding sites and direct target genes of a floral master regulatory transcription factor [RNA-Seq

    NARCIS (Netherlands)

    Muiño, J.M.; Bruijn, de S.A.; Vingron, Martin; Angenent, G.C.; Kaufmann, Kerstin

    2015-01-01

    Plant development is controlled by transcription factors (TFs) which form complex gene-regulatory networks. Genome-wide TF DNA-binding studies revealed that these TFs have several thousands of binding sites in the Arabidopsis genome, and may regulate the expression of many genes directly. Given the

  2. Sexual selection at the protein level drives the extraordinary divergence of sex-related genes during sympatric speciation

    NARCIS (Netherlands)

    Van Doorn, G.S.; Luttikhuizen, P.C; Weissing, F.J.

    2001-01-01

    An increasing number of molecular studies are indicating that, in a wide variety of species, genes directly related to fertilization evolve at extraordinarily high rates. We try to gain insight into the dynamics of this rapid evolution and its underlying mechanisms by means of a simple theoretical

  3. Solvent Effects on Oxygen-17 Chemical Shifts in Amides. Quantitative Linear Solvation Shift Relationships

    Science.gov (United States)

    Díez, Ernesto; Fabián, Jesús San; Gerothanassis, Ioannis P.; Esteban, Angel L.; Abboud, José-Luis M.; Contreras, Ruben H.; de Kowalewski, Dora G.

    1997-01-01

    A multiple-linear-regression analysis (MLRA) has been carried out using the Kamlet-Abboud-Taft (KAT) solvatochromic parameters in order to elucidate and quantify the solvent effects on the17O chemical shifts ofN-methylformamide (NMF),N,N-dimethylformamide (DMF),N-methylacetamide (NMA), andN,N-dimethylacetamide (DMA). The chemical shifts of the four molecules show the same dependence (in ppm) on the solvent polarity-polarizability, i.e., -22π*. The influence of the solvent hydrogen-bond-donor (HBD) acidities is slightly larger for the acetamides NMA and DMA, i.e., -48α, than for the formamides NMF and DMF, i.e., -42α. The influence of the solvent hydrogen-bond-acceptor (HBA) basicities is negligible for the nonprotic molecules DMF and DMA but significant for the protic molecules NMF and NMA, i.e., -9β. The effect of substituting the N-H hydrogen by a methyl group amounts to -5.9 ppm in NMF and 5.4 ppm in NMA. The effect of substituting the O=C-H hydrogen amounts to 5.5 ppm in NMF and 16.8 ppm in DMF. The model of specific hydration sites of amides by I. P. Gerothanassis and C. Vakka [J. Org. Chem.59,2341 (1994)] is settled in a more quantitative basis and the model by M. I. Burgar, T. E. St. Amour, and D. Fiat [J. Phys. Chem.85,502 (1981)] is critically evaluated.17O hydration shifts have been calculated for formamide (FOR) by the ab initio LORG method at the 6-31G* level. For a formamide surrounded by the four in-plane molecules of water in the first hydration shell, the calculated17O shift change due to the four hydrogen bonds, -83.2 ppm, is smaller than the empirical hydration shift, -100 ppm. The17O shift change from each out-of-plane water molecule hydrogen-bonded to the amide oxygen is -18.0 ppm. These LORG results support the conclusion that no more than four water molecules are hydrogen-bonded to the amide oxygen in formamide.

  4. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2016-09-01

    Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

  5. Evaluation of clinical sensitivity and specificity of hepatitis B virus (HBV), hepatitis C virus, and human immunodeficiency Virus-1 by cobas MPX: Detection of occult HBV infection in an HBV-endemic area.

    Science.gov (United States)

    Ha, Jihye; Park, Younhee; Kim, Hyon-Suk

    2017-11-01

    Transfusion-transmitted infectious diseases remain a major concern for blood safety, particularly with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Nucleic acid testing (NAT) in donor screening shortens the serologically negative window period and reduces virus transmission. The cobas MPX (Roche Molecular Systems, Inc., Branchburg, New Jersey) is a recently developed multiplex qualitative PCR system that enables the simultaneous detection of HBV, HCV, and HIV with improved sensitivity and throughput using cobas 6800 and 8800 instruments. The aim of this study was to conduct an evaluation of the clinical sensitivity and specificity of cobas MPX detection of HBV, HCV, and HIV in clinical specimens. Among samples referred for HBV, HCV, and HIV-1 quantification at Severance Hospital, Yonsei University College of Medicine, positive samples were selected to evaluate sensitivity. A total of 843 samples was tested using both cobas MPX and COBAS AmpliPrep/COBAS TaqMan Tests for HBV, HCV, and HIV-1 using the cobas 8800 system and a COBAS TaqMan 96 analyzer, respectively. Samples that showed discrepancies were confirmed by nested PCR. The cobas MPX achieved excellent sensitivity and specificity for the detection of HBV, HCV, and HIV-1 in clinical samples. We found that the lower limit of detection (LOD) of blood screening by NAT actually improves clinical sensitivity, and occult HBV infection prevalence among healthy employees of the hospital was rather high. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-11-01

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

  7. Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

    Science.gov (United States)

    Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

    2016-03-01

    HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Hypothalamus transcriptome profile suggests an anorexia-cachexia syndrome in the anx/anx mouse model.

    Science.gov (United States)

    Mercader, Josep Maria; Lozano, Juan José; Sumoy, Lauro; Dierssen, Mara; Visa, Joana; Gratacòs, Mònica; Estivill, Xavier

    2008-11-12

    The anx/anx mouse displays poor appetite and lean appearance and is considered a good model for the study of anorexia nervosa. To identify new genes involved in feeding behavior and body weight regulation we performed an expression profiling in the hypothalamus of the anx/anx mice. Using commercial microarrays we detected 156 differentially expressed genes and validated 92 of those using TaqMan low-density arrays. The expression of a set of 87 candidate genes selected based on literature evidences was also quantified by TaqMan low-density arrays. Our results showed enrichment in deregulated genes involved in cell death, cell morphology, and cancer, as well as an alteration of several signaling circuits involved in energy balance including neuropeptide Y and melanocortin signaling. The expression profile along with the phenotype led us to conclude that anx/anx mice resemble the anorexia-cachexia syndrome typically observed in cancer, infection with human immunodeficiency virus or chronic diseases, rather than starvation, and that anx/anx mice could be considered a good model for the treatment and investigation of this condition.

  9. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    Science.gov (United States)

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Circumvention of normal constraints on granule protein gene expression in peripheral blood neutrophils and monocytes of patients with antineutrophil cytoplasmic autoantibody-associated glomerulonephritis.

    Science.gov (United States)

    Yang, Jia Jin; Pendergraft, William F; Alcorta, David A; Nachman, Patrick H; Hogan, Susan L; Thomas, Robin P; Sullivan, Pamela; Jennette, J Charles; Falk, Ronald J; Preston, Gloria A

    2004-08-01

    Granulopoiesis-related genes are distinctively upregulated in peripheral leukocytes of patients with antineutrophil cytoplasmic autoantibodies (ANCA)-associated glomerulonephritis. Affymetrix microarrays identified the upregulation of nine neutrophilic primary granule genes, including myeloperoxidase (MPO) and proteinase 3 (PR3), plus five secondary granule genes. Coordinate expression of granulocyte maturation marker CD35, measured by TaqMan PCR, and positive in situ staining for PR3 transcripts in polymorphic neutrophils and monocytes indicate that these genes are expressed in "mature" cells. Increased transcripts correlated with disease activity and absolute neutrophil values but not with "left shift," drug regimen, cytokine levels, hematuria, proteinuria, ANCA titer, serum creatinine, gender, or age. Upregulation of PR3 and MPO transcripts was specifically associated with ANCA disease (n = 56) as these changes were not detected in patients with ESRD (n = 25) or systemic lupus erythematosus (n = 17), as determined by TaqMan PCR. This is the first report of this phenomenon in nonneoplastic cells. The data raise the hypothesis that, in addition to the presence of anti-MPO or anti-PR3 autoantibodies, a second critical component in the cause of this disease is the reactivation of once-silenced genes leading to increased antigen availability.

  11. RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature

    Science.gov (United States)

    Viollet, Coralie; Davis, David A.; Tekeste, Shewit S.; Reczko, Martin; Pezzella, Francesco; Ragoussis, Jiannis

    2017-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases. PMID:28046107

  12. Development of real-time PCR assay for genetic identification of the mottled skate, Beringraja pulchra.

    Science.gov (United States)

    Hwang, In Kwan; Lee, Hae Young; Kim, Min-Hee; Jo, Hyun-Su; Choi, Dong-Ho; Kang, Pil-Won; Lee, Yang-Han; Cho, Nam-Soo; Park, Ki-Won; Chae, Ho Zoon

    2015-10-01

    The mottled skate, Beringraja pulchra is one of the commercially important fishes in the market today. However, B. pulchra identification methods have not been well developed. The current study reports a novel real-time PCR method based on TaqMan technology developed for the genetic identification of B. pulchra. The mitochondrial cytochrome oxidase subunit 1 (COI) nucleotide sequences of 29 B. pulchra, 157 skates and rays reported in GenBank DNA database were comparatively analyzed and the COI sequences specific to B. pulchra was identified. Based on this information, a system of specific primers and Minor Groove Binding (MGB) TaqMan probe were designed. The assay successfully discriminated in 29 specimens of B. pulchra and 27 commercial samples with unknown species identity. For B. pulchra DNA, an average Threshold Cycle (Ct) value of 19.1±0.1 was obtained. Among 27 commercial samples, two samples showed average Ct values 19.1±0.0 and 26.7±0.1, respectively and were confirmed to be B. pulchra based on sequencing. The other samples tested showed undetectable or extremely weak signals for the target fragment, which was also consistent with the sequencing results. These results reveal that the method developed is a rapid and efficient tool to identify B. pulchra and might prevent fraud or mislabeling during the distribution of B. pulchra products. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Rapid Detection of the Chlamydiaceae and Other Families in the Order Chlamydiales: Three PCR Tests

    Science.gov (United States)

    Everett, Karin D. E.; Hornung, Linda J.; Andersen, Arthur A.

    1999-01-01

    Few identification methods will rapidly or specifically detect all bacteria in the order Chlamydiales, family Chlamydiaceae. In this study, three PCR tests based on sequence data from over 48 chlamydial strains were developed for identification of these bacteria. Two tests exclusively recognized the Chlamydiaceae: a multiplex test targeting the ompA gene and the rRNA intergenic spacer and a TaqMan test targeting the 23S ribosomal DNA. The multiplex test was able to detect as few as 200 inclusion-forming units (IFU), while the TaqMan test could detect 2 IFU. The amplicons produced in these tests ranged from 132 to 320 bp in length. The third test, targeting the 23S rRNA gene, produced a 600-bp amplicon from strains belonging to several families in the order Chlamydiales. Direct sequence analysis of this amplicon has facilitated the identification of new chlamydial strains. These three tests permit ready identification of chlamydiae for diagnostic and epidemiologic study. The specificity of these tests indicates that they might also be used to identify chlamydiae without culture or isolation. PMID:9986815

  14. Identification of four squid species by quantitative real-time polymerase chain reaction.

    Science.gov (United States)

    Ye, Jian; Feng, Junli; Liu, Shasha; Zhang, Yanping; Jiang, Xiaona; Dai, Zhiyuan

    2016-02-01

    Squids are distributed worldwide, including many species of commercial importance, and they are often made into varieties of flavor foods. The rapid identification methods for squid species especially their processed products, however, have not been well developed. In this study, quantitative real-time PCR (qPCR) systems based on specific primers and TaqMan probes have been established for rapid and accurate identification of four common squid species (Ommastrephes bartramii, Dosidicus gigas, Illex argentinus, Todarodes pacificus) in Chinese domestic market. After analyzing mitochondrial genes reported in GenBank, the mitochondrial cytochrome b (Cytb) gene was selected for O. bartramii detection, cytochrome c oxidase subunit I (COI) gene for D. gigas and T. Pacificus detection, ATPase subunit 6 (ATPase 6) gene for I. Argentinus detection, and 12S ribosomal RNA (12S rDNA) gene for designing Ommastrephidae-specific primers and probe. As a result, all the TaqMan systems are of good performance, and efficiency of each reaction was calculated by making standard curves. This method could detect target species either in single or mixed squid specimen, and it was applied to identify 12 squid processed products successfully. Thus, it would play an important role in fulfilling labeling regulations and squid fishery control. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. A new QRT-PCR assay designed for the differentiation between elements provided from Agrobacterium sp. in GMOs plant events and natural Agrobacterium sp. bacteria.

    Science.gov (United States)

    Nabi, Nesrine; Chaouachi, Maher; Zellama, Mohamed Salem; Ben Hafsa, Ahmed; Mrabet, Besma; Saïd, Khaled; Fathia, Harzallah Skhiri

    2016-04-01

    The question asked in the present work was how to differentiate between contamination of field samples with and GM plants contained sequences provided from this bacterium in order to avoid false positives in the frame of the detection and the quantification of GMO. For this, new set of primers and corresponding TaqMan Minor Groove Binder (MGB) probes were designed to target Agrobacterium sp. using the tumor-morphology-shooty gene (TMS1). Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log (colony forming units) per milliliter) via linear regression. The method designed was highly specific and sensitive, with a detection limit of 10CFU/ml. No significant cross-reaction was observed. Results from this study showed that TaqMan real-time PCR, is potentially an effective method for the rapid and reliable quantification of Agrobacterium sp. in samples containing GMO or non GMO samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Development and application of a universal Hemoplasma screening assay based on the SYBR green PCR principle.

    Science.gov (United States)

    Willi, Barbara; Meli, Marina L; Lüthy, Ruedi; Honegger, Hanspeter; Wengi, Nicole; Hoelzle, Ludwig E; Reusch, Claudia E; Lutz, Hans; Hofmann-Lehmann, Regina

    2009-12-01

    Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.

  17. Development of a spreadsheet for SNPs typing using Microsoft EXCEL.

    Science.gov (United States)

    Hashiyada, Masaki; Itakura, Yukio; Takahashi, Shirushi; Sakai, Jun; Funayama, Masato

    2009-04-01

    Single-nucleotide polymorphisms (SNPs) have some characteristics that make them very appropriate for forensic studies and applications. In our institute, SNPs typings were performed by the TaqMan SNP Genotyping Assays using the ABI PRISM 7500 FAST Real-Time PCR System (AppliedBiosystems) and Sequence Detection Software ver.1.4 (AppliedBiosystem). The TaqMan method was desired two positive control (Allele1 and 2) and one negative control to analyze each SNP locus. Therefore, it can be analyzed up to 24 loci of a person on a 96-well-plate at the same time. If SNPs analysis is expected to apply to biometrics authentication, 48 and over loci are required to identify a person. In this study, we designed a spreadsheet package using Microsoft EXCEL, and population data were used from our 120 SNPs population studies. On the spreadsheet, we defined SNP types using 'template files' instead of positive and negative controls. "Template files" consisted of the results of 94 unknown samples and two negative controls of each of 120 SNPs loci we had previously studied. By the use of the files, the spreadsheet could analyze 96 SNPs on a 96-wells-plate simultaneously.

  18. Detection of knockdown resistance (kdr mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    Directory of Open Access Journals (Sweden)

    Ball Amanda

    2007-08-01

    Full Text Available Abstract Background Knockdown resistance (kdr is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1 TaqMan probes and 2 high resolution melt (HRM analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR, Heated Oligonucleotide Ligation Assay (HOLA, Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost, and safety (requirement for hazardous chemicals. Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions and the most specific (with the lowest number of incorrect scores. Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS

  19. Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

    Science.gov (United States)

    Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

    2017-07-01

    Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for

  20. Melt analysis of mismatch amplification mutation assays (Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models.

    Directory of Open Access Journals (Sweden)

    Dawn N Birdsell

    Full Text Available Single nucleotide polymorphisms (SNPs are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA, is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg. Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of

  1. Development of a multiplex assay for genus- and species-specific detection of Phytophthora based on differences in mitochondrial gene order.

    Science.gov (United States)

    Bilodeau, Guillaume J; Martin, Frank N; Coffey, Michael D; Blomquist, Cheryl L

    2014-07-01

    A molecular diagnostic assay for Phytophthora spp. that is specific, sensitive, has both genus- and species-specific detection capabilities multiplexed, and can be used to systematically develop markers for detection of a wide range of species would facilitate research and regulatory efforts. To address this need, a marker system was developed based on the high copy sequences of the mitochondrial DNA utilizing gene orders that were highly conserved in the genus Phytophthora but different in the related genus Pythium and plants to reduce the importance of highly controlled annealing temperatures for specificity. An amplification primer pair designed from conserved regions of the atp9 and nad9 genes produced an amplicon of ≈340 bp specific for the Phytophthora spp. tested. The TaqMan probe for the genus-specific Phytophthora test was designed from a conserved portion of the atp9 gene whereas variable intergenic spacer sequences were used for designing the species-specific TaqMan probes. Specific probes were developed for 13 species and the P. citricola species complex. In silico analysis suggests that species-specific probes could be developed for at least 70 additional described and provisional species; the use of locked nucleic acids in TaqMan probes should expand this list. A second locus spanning three tRNAs (trnM-trnP-trnM) was also evaluated for genus-specific detection capabilities. At 206 bp, it was not as useful for systematic development of a broad range of species-specific probes as the larger 340-bp amplicon. All markers were validated against a test panel that included 87 Phytophthora spp., 14 provisional Phytophthora spp., 29 Pythium spp., 1 Phytopythium sp., and 39 plant species. Species-specific probes were validated further against a range of geographically diverse isolates to ensure uniformity of detection at an intraspecific level, as well as with other species having high levels of sequence similarity to ensure specificity. Both diagnostic

  2. Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay

    Directory of Open Access Journals (Sweden)

    Rupreht Ruth

    2007-11-01

    Full Text Available Abstract Background Hereditary hemochromatosis (HH is a common genetic disease characterized by excessive iron overload that leads to multi-organ failure. Although the most prevalent genotype in HH is homozygosity for C282Y mutation of the HFE gene, two additional mutations, H63D and S65C, appear to be associated with a milder form of HH. The aim of this study was to develop a high-throughput assay for HFE mutations screening based on TaqMan technology and to determine the frequencies of HFE mutations in the Slovenian population. Methods Altogether, 1282 randomly selected blood donors from different Slovenian regions and 21 HH patients were analyzed for the presence of HFE mutations by an in-house developed real-time PCR assay based on TaqMan technology using shorter non-interfering fluorescent single nucleotide polymorphism (SNP-specific MGB probes. The assay was validated by RFLP analysis and DNA sequencing. Results The genotyping assay of the H63D, S65C and C282Y mutations in the HFE gene, based on TaqMan technology proved to be fast, reliable, with a high-throughput capability and 100% concordant with genotypes obtained by RFLP and DNA sequencing. The observed frequency of C282Y homozygotes in the group of HH patients was only 48%, others were of the heterogeneous HFE genotype. Among 1282 blood donors tested, the observed H63D, S65C and C282Y allele frequency were 12.8% (95% confidence interval (CI 11.5 – 14.2%, 1.8% (95% CI 1.4 – 2.5% and 3.6% (95% CI 3.0 – 4.5%, respectively. Approximately 33% of the tested subjects had at least one of the three HH mutations, and 1% of them were C282Y homozygotes or compound heterozygotes C282Y/H63D or C282Y/S65C, presenting an increased risk for iron overload disease. A significant variation in H63D allele frequency was observed for one of the Slovenian regions. Conclusion The improved real-time PCR assay for H63D, S65C and C282Y mutations detection is accurate, fast, cost-efficient and ready for

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

    Science.gov (United States)

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

    2017-02-07

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

  4. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  5. Diagnostic efficacy of a real time-PCR assay for Chlamydia trachomatis infection in infertile women in north India

    Directory of Open Access Journals (Sweden)

    Benu Dhawan

    2014-01-01

    Full Text Available Background & objectives: Little is known about the prevalence of Chlamydia trachomatis infection in Indian women with infertility. To improve the diagnosis of C. trachomatis infection in developing countries, there is an urgent need to establish cost-effective molecular test with high sensitivity and specificity. This study was conducted to determine the diagnostic utility of a real time-PCR assay for detention of C. trachomatis infection in infertile women attending an infertility clinic in north India. The in house real time-PCR assay was also compared with a commercial real-time PCR based detection system. Methods: Endocervical swabs, collected from 200 infertile women were tested for C. trachomatis by three different PCR assays viz. in-house real time-PCR targeting the cryptic plasmid using published primers, along with omp1 gene and cryptic plasmid based conventional PCR assays. Specimens were also subjected to direct fluorescence assay (DFA and enzyme immunoassay (EIA Performance of in-house real time-PCR was compared with that of COBAS Taqman C. trachomatis Test, version 2.0 on all in-house real time-PCR positive sample and 30 consecutive negative samples. Results: C. trachomatis infection was found in 13.5 per cent (27/200 infertile women by in-house real time-PCR, 11.5 per cent (23/200 by cryptic plasmid and/or omp1 gene based conventional PCR, 9 per cent (18/200 by DFA and 6.5 per cent (7/200 by EIA. The in-house real time-PCR exhibited a sensitivity and specificity of 100 per cent, considering COBAS Taqman CT Test as the gold standard. The negative and positive predictive values of the in-house real time-PCR were 100 per cent. The in-house real time-PCR could detect as low as 10 copies of C. trachomatis DNA per reaction. Interpretation & conclusions: In-house real time-PCR targeting the cryptic plasmid of C. trachomatis exhibited an excellent sensitivity and specificity similar to that of COBAS Taqman CT Test, v2.0 for detection of C

  6. Repeat testing of low-level HIV-1 RNA: assay performance and implementation in clinical trials.

    Science.gov (United States)

    White, Kirsten; Garner, Will; Wei, Lilian; Eron, Joseph J; Zhong, Lijie; Miller, Michael D; Martin, Hal; Plummer, Andrew; Tran-Muchowski, Cecilia; Lindstrom, Kim; Porter, James; Piontkowsky, David; Light, Angela; Reiske, Heinz; Quirk, Erin

    2018-05-15

    Assess the performance of HIV-1 RNA repeat testing of stored samples in cases of low-level viremia during clinical trials. Prospective and retrospective analysis of randomized clinical trial samples and reference standards. To evaluate assay variability of the Cobas AmpliPrep/Cobas TaqMan HIV-1 Test, v2.0, three separate sources of samples were utilized: the World Health Organization (WHO) HIV reference standard (assayed using 50 independent measurements at six viral loads <200 copies/ml), retrospective analysis of four to six aliquots of plasma samples from four clinical trial participants, and prospective repeat testing of 120 samples from participants in randomized trials with low-level viremia. The TaqMan assay on the WHO HIV-1 RNA standards at viral loads <200 copies/ml performed within the expected variability according to assay specifications. However, standards with low viral loads of 36 and 18 copies/ml reported values of ≥ 50 copies/ml in 66 and 18% of tests, respectively. In participants treated with antiretrovirals who had unexpected viremia of 50-200 copies/ml after achieving <50 copies/ml, retesting of multiple aliquots of stored plasma found <50 copies/ml in nearly all cases upon retesting (14/15; 93%). Repeat testing was prospectively implemented in four clinical trials for all samples with virologic rebound of 50-200 copies/ml (n = 120 samples from 92 participants) from which 42% (50/120) had a retest result of less than 50 copies/ml and 58% (70/120) retested ≥ 50 copies/ml. The TaqMan HIV-1 RNA assay shows variability around 50 copies/ml that affects clinical trial results and may impact clinical practice. In participants with a history of viral load suppression, unexpected low-level viremia may be because of assay variability rather than low drug adherence or true virologic failure. Retesting a stored aliquot of the same sample may differentiate between assay variability and virologic failure as the source of viremia

  7. Clinical evaluation of a quantitative real time polymerase chain reaction assay for diagnosis of primary Epstein-Barr virus infection in children.

    Science.gov (United States)

    Pitetti, Raymond D; Laus, Stella; Wadowsky, Robert M

    2003-08-01

    Epstein-Barr virus (EBV) infectious mononucleosis is often diagnosed based on characteristic clinical features and either a positive heterophil antibody test or serology, both of which can be unreliable in young children. Real time quantitative PCR assays that measure EBV DNA load in serum or plasma are highly sensitive in young children, but serum and plasma contain inhibitors of PCR which must be removed by DNA extraction techniques. A real time TaqMan PCR assay was designed and evaluated for simultaneously measuring EBV DNA load and validating the removal of PCR inhibitors from serum samples. A serum sample was available from patients classified serologically as primary EBV infection (n = 28), EBV-seronegative (n = 25) and EBV-seropositive (n = 26). Patients were classified as having EBV infectious mononucleosis if they had specified clinical findings and > or =10% atypical lymphocytes in peripheral blood or had a positive Monospot test result. DNA was purified by a spin column method and tested in PCR reactions with primers for EBV DNA polymerase gene and internal control targets. Amplification of the two PCR products was measured in real time with separate TaqMan DNA probes labeled with various fluorescent reporters. The mean age of study patients was 9 years, 4 months. Twenty-one (75%) of the patients in the primary EBV infection group, one (4%) of the seronegatives and none of the seropositives had detectable EBV DNA. Within the primary infection group, those with detectable virus were more likely than those without detectable virus to have evidence of lymphadenopathy (14 of 16 vs.1 of 5; P = 0.011), higher mean atypical (11.7 vs.0.9%; P = 0.002) and absolute atypical (1.5 vs.0.1 x 109/l; P = 0.004) lymphocyte count, higher mean absolute lymphocyte count (4.7 vs.2.3 x 109/l; P = 0.026) and higher mean aspartate aminotransferase value (119.8 vs.37.3 IU/l; P = 0.036). Ten patients, all in the primary infection group, had EBV infectious mononucleosis, and all

  8. Molecular methods for typing of Helicobacter pylori and their applications

    DEFF Research Database (Denmark)

    Colding, H; Hartzen, S H; Roshanisefat, H

    1999-01-01

    .g. the urease genes. Furthermore, reproducibility, discriminatory power, ease of performance and interpretation, cost and toxic procedures of each method are assessed. To date no direct comparison of all the molecular typing methods described has been performed in the same study with the same H. pylori strains....... However, PCR analysis of the urease gene directly on suspensions of H. pylori or gastric biopsy material seems to be useful for routine use and applicable in specific epidemiological situations....

  9. Deficit of Kcnma1 mRNA expression in the dentate gyrus of epileptic rats

    Science.gov (United States)

    Ermolinsky, Boris; Arshadmansab, Massoud F.; Pacheco Otalora, Luis F.; Zarei, Masoud M.; Garrido-Sanabria, Emilio R.

    2008-01-01

    Epileptogenesis in mesial temporal lobe epilepsy is determined by several factors including abnormalities in the expression and function of ion channels. Here, we report a long-lasting deficit in gene expression of Kcnma1 coding for the large-conductance calcium-activated potassium (BK, MaxiK) channel α-subunits after pilocarpine-induced status epilepticus. By using comparative real-time PCR, Taqman gene expression assays, and the delta-delta comparative threshold method we detected a significant reduction in Kcnma1 expression in microdissected dentate gyrus at different intervals after status epilepticus (24 h, 10 days, 1 month, and more than 2 months). BK channels are key regulators of neuronal excitability and transmitter release. Hence, defective Kcnma1 expression may play a critical role in the pathogenesis of mesial temporal lobe epilepsy. PMID:18695509

  10. Lack of an association of miR-938 SNP in IDDM10 with human type 1 diabetes

    Directory of Open Access Journals (Sweden)

    Mi Xiaofan

    2011-10-01

    Full Text Available Abstract MicroRNAs (miRNAs are a newly discovered type of small non-protein coding RNA that function in the inhibition of effective mRNA translation, and may serve as susceptibility genes for various disease developments. The SNP rs12416605, located in human type 1 diabetes IDDM10 locus, changes the seeding sequence (UGU[G/A]CCC of miRNA miR-938 and potentially alters miR-938 targets, including IL-16 and IL-17A. In an attempt to test whether miR-938 may be a susceptibility gene for IDDM10, we assessed the possible association of the miR-938 SNP with T1D in an American Caucasian cohort of 622 patients and 723 healthy controls by TaqMan assay. Our current data do not support the association between the SNP in miR-938 and type 1 diabetes.

  11. 8q24 sequence variants in relation to prostate cancer risk among men of African descent: A case-control study

    Directory of Open Access Journals (Sweden)

    VanCleave Tiva T

    2010-06-01

    Full Text Available Abstract Background Human chromosome 8q24 has been implicated in prostate tumorigenesis. Methods Consequently, we evaluated seven 8q24 sequence variants relative to prostate cancer (PCA in a case-control study involving men of African descent. Genetic alterations were detected in germ-line DNA from 195 incident PCA cases and 531 controls using TaqMan polymerase chain reaction (PCR. Results Inheritance of the 8q24 rs16901979 T allele corresponded to a 2.5-fold increase in the risk of developing PCA for our test group. These findings were validated using multifactor dimensionality reduction (MDR and permutation testing (p = 0.038. The remaining 8q24 targets were not significantly related to PCA outcomes. Conclusions Although compelling evidence suggests that the 8q24 rs16901979 locus may serve as an effective PCA predictor, our findings require additional evaluation in larger studies.

  12. Nucleolin is regulated both at the level of transcription and translation

    International Nuclear Information System (INIS)

    Bicknell, Katrina; Brooks, Gavin; Kaiser, Pete; Chen Hongying; Dove, Brian K.; Hiscox, Julian A.

    2005-01-01

    Nucleolin is a multi-functional protein that is located to the nucleolus. In tissue culture cells, the stability of nucleolin is related to the proliferation status of the cell. During development, rat cardiomyocytes proliferate actively with increases in the mass of the heart being due to both hyperplasia and hypertrophy. The timing of this shift in the phenotype of the myocyte from one capable of undergoing hyperplasia to one that can grow only by hypertrophy occurs within 4 days of post-natal development. Thus, cardiomyocytes are an ideal model system in which to study the regulation of nucleolin during growth in vivo. Using Western blot and quantitative RT-PCR (TaqMan) we found that the amount of nucleolin is regulated both at the level of transcription and translation during the development of the cardiomyocyte. However, in cells which had exited the cell cycle and were subsequently given a hypertrophic stimulus, nucleolin was regulated post-transcriptionally

  13. Studies of CTNNBL1 and FDFT1 variants and measures of obesity: analyses of quantitative traits and case-control studies in 18,014 Danes

    DEFF Research Database (Denmark)

    Andreasen, Camilla Helene; Mogensen, Mette Sloth; Borch-Johnsen, Knut

    2009-01-01

    of obesity-related quantitative traits, and case-control studies in large study samples of Danes. METHODS: The FDFT1 rs7001819, CTNNBL1 rs6013029 and rs6020846 were genotyped, using TaqMan allelic discrimination, in a combined study sample comprising 18,014 participants ascertained from; the population...... and a previous study. FDFT1 rs7001819 showed no association with obesity, neither when analysing quantitative traits nor when performing case-control studies of obesity.......). The most significantly associating variants within CTNNBL1 including rs6013029 and rs6020846 were additionally confirmed to associate with morbid obesity in a French Caucasian case-control sample. The aim of this study was to investigate the impact of these three variants on obesity, through analyses...

  14. A random PCR screening system for the identification of type 1 human herpes simplex virus.

    Science.gov (United States)

    Yu, Xuelian; Shi, Bisheng; Gong, Yan; Zhang, Xiaonan; Shen, Silan; Qian, Fangxing; Gu, Shimin; Hu, Yunwen; Yuan, Zhenghong

    2009-10-01

    Several viral diseases exhibit measles-like symptoms. Differentiation of suspected cases of measles with molecular epidemiological techniques in the laboratory is useful for measles surveillance. In this study, a random PCR screening system was undertaken for the identification of isolates from patients with measles-like symptoms who exhibited cytopathic effects, but who had negative results for measles virus-specific reverse transcription (RT)-PCR and indirect immunofluorescence assays. Sequence analysis of random amplified PCR products showed that they were highly homologous to type 1 human herpes simplex virus (HSV-1). The results were further confirmed by an HSV-1-specific TaqMan real-time PCR assay. The random PCR screening system described in this study provides an efficient procedure for the identification of unknown viral pathogens. Measles-like symptoms can also be caused by HSV-1, suggesting the need to include HSV-1 in differential diagnoses of measles-like diseases.

  15. The GCKR rs780094 polymorphism is associated with elevated fasting serum triacylglycerol, reduced fasting and OGTT-related insulinaemia, and reduced risk of type 2 diabetes

    DEFF Research Database (Denmark)

    Sparsø, T; Andersen, G; Nielsen, T

    2007-01-01

    to metabolic traits (mainly fasting hypertriacylglycerolaemia) and traits related to pancreatic beta cell function. METHODS: The polymorphism was genotyped in 16,853 Danes using Taqman allelic discrimination. Association was analysed in case-control studies and quantitative trait analyses. We also analysed...... the possible interactive effect between the GCK -30G>A polymorphism and the GCKR rs780094 variant on metabolic traits. RESULTS: The minor GCKR A-allele of rs780094 is associated with an increased level of fasting serum triacylglycerol (p = 6 x 10(-14)), impaired fasting (p = 0.001) and OGTT-related insulin...... release (p = 3 x 10(-6)), reduced homeostasis model assessment of insulin resistance (p = 0.0004), WHO-defined dyslipidaemia (p = 6 x 10(-9)) and a modestly decreased risk of type 2 diabetes (p = 0.01). Significantly increased fasting serum insulin concentrations were demonstrated when analysing the GCK...

  16. Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes

    DEFF Research Database (Denmark)

    Schepeler, Troels; Holm, Anja; Halvey, P

    2012-01-01

    Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array......RNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes....... MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy...

  17. MAVS is not a Likely Susceptibility Locus for Addison's Disease and Type 1 Diabetes.

    Science.gov (United States)

    Zurawek, Magdalena; Fichna, Marta; Kazimierska, Marta; Fichna, Piotr; Dzikiewicz-Krawczyk, Agnieszka; Przybylski, Grzegorz; Ruchala, Marek; Nowak, Jerzy

    2017-06-01

    Mitochondrial antiviral signaling (MAVS) protein is an intracellular adaptor molecule, downstream of viral sensors, retinoid acid-inducible gene I (RIG-I)-like receptors (RLRs). Impaired antiviral cell signaling might contribute to autoimmunity. Studies have recently shown variations in genes encoding RLRs as risk factors for autoimmune diseases. We investigated whether MAVS coding polymorphisms are associated with Addison's disease (AD) and type 1 diabetes (T1D) in Polish population. We genotyped 140 AD, 532 T1D patients and 600 healthy controls for MAVS rs17857295, rs7262903, rs45437096 and rs7269320. Genotyping was performed by TaqMan assays. Distribution of the MAVS genotypes and alleles did not reveal significant differences between patients and controls (p > 0.05). This analysis did not indicate the association of the MAVS locus with susceptibility to AD and T1D.

  18. SCN1A, ABCC2 and UGT2B7 gene polymorphisms in association with individualized oxcarbazepine therapy.

    Science.gov (United States)

    Ma, Chun-Lai; Wu, Xun-Yi; Jiao, Zheng; Hong, Zhen; Wu, Zhi-Yuan; Zhong, Ming-Kang

    2015-01-01

    Associations between the effects of SCN1A, SCN2A, ABCC2 and UGT2B7 genetic polymorphisms and oxcarbazepine (OXC) maintenance doses in Han Chinese epileptic patients were investigated. Genetic polymorphisms were detected in 184 epileptic patients receiving OXC monotherapy by high-resolution melting curve and TaqMan method. Carriers of the SCN1A IVS5-91G>A, UGT2B7 c.802T>C and ABCC2 c.1249G>A variant alleles required significantly higher OXC maintenance doses than noncarriers (p GA > AA. SCN1A, UGT2B7 and ABCC2 genetic polymorphisms are associated with OXC maintenance doses and may be useful for the personalization of OXC therapy in epileptic patients. Further studies are needed. Original submitted 6 June 2014; Revision submitted 5 September 2014.

  19. Age and gender differences in the influences of eNOS T-786C polymorphism on arteriosclerotic parameters in general population in Japan.

    Science.gov (United States)

    Hashimoto, Marowa; Miyai, Nobuyuki; Hattori, Sonomi; Iwahara, Akihiko; Utsumi, Miyoko; Arita, Mikio; Takeshita, Tatsuya

    2016-07-01

    The influence of T-786C polymorphism in the promoter region of endothelial nitric oxide synthase (eNOS) on arteriosclerotic parameters by age and gender were examined. Brachial-ankle pulse wave velocity (baPWV), heart-rate adjusted augmentation index (AIx@75), pulse pressure (PP) and albumin-creatinine ratio (ACR) were assessed as arteriosclerotic parameters in addition to non-high-density lipoprotein cholesterol (non-HDL-C) to HDL-C (non-HDL-C/HDL-C) ratio in 1499 participants. T-786C polymorphism (rs2070744) was screened using a TaqMan allelic discrimination assay. Analyses of covariance were carried. Women with the non-C allele showed significantly lower AIx@75 in participants aged gender differences, possibly involving antioxidative and/or endothelial signaling other than inflammatory signaling.

  20. BRIP1 (BACH1 variants and familial breast cancer risk: a case-control study

    Directory of Open Access Journals (Sweden)

    Bugert Peter

    2007-05-01

    Full Text Available Abstract Background Inactivating and truncating mutations of the nuclear BRCA1-interacting protein 1 (BRIP1 have been shown to be the major cause of Fanconi anaemia and, due to subsequent alterations of BRCA1 function, predispose to breast cancer (BC. Methods We investigated the effect of BRIP1 -64G>A and Pro919Ser on familial BC risk by means of TaqMan allelic discrimination, analysing BRCA1/BRCA2 mutation-negative index patients of 571 German BC families and 712 control individuals. Results No significant differences in genotype frequencies between BC cases and controls for BRIP1 -64G>A and Pro919Ser were observed. Conclusion We found no effect of the putatively functional BRIP1 variants -64G>A and Pro919Ser on the risk of familial BC.

  1. Direct detection of Leishmania from clinical samples.

    Science.gov (United States)

    Waitumbi, John N; Bast, Joshua; Nyakoe, Nancy; Magiri, Charles; Quintana, Miguel; Takhampunya, Ratree; Schuster, Anthony L; Van de Wyngaerde, Marshall T; McAvin, James C; Coleman, Russell E

    2017-01-01

    The ability to rapidly and accurately diagnose leishmaniasis is a military priority. Testing was conducted to evaluate diagnostic sensitivity and specificity of field-expedient Leishmania genus and visceral Leishmania specific dual-fluorogenic, hydrolysis probe (TaqMan), polymerase chain reaction assays previously established for use in vector surveillance. Blood samples of patients with confirmed visceral leishmaniasis and controls without the disease from Baringo District, Kenya, were tested. Leishmania genus assay sensitivity was 100% (14/14) and specificity was 84% (16/19). Visceral Leishmania assay sensitivity was 93% (13/14) and specificity 80% (4/5). Cutaneous leishmaniasis (CL) skin scrapes of patients from Honduras were also evaluated. Leishmania genus assay sensitivity was 100% (10/10). Visceral Leishmania assay specificity was 100% (10/10) from cutaneous leishmaniasis samples; no fluorescence above background was reported. These results show promise in a rapid, sensitive, and specific method for Leishmania direct detection from clinical samples.

  2. Comparative analysis of minor histocompatibility antigens genotyping methods

    Directory of Open Access Journals (Sweden)

    A. S. Vdovin

    2016-01-01

    Full Text Available The wide range of techniques could be employed to find mismatches in minor histocompatibility antigens between transplant recipients and their donors. In the current study we compared three genotyping methods based on polymerase chain reaction (PCR for four minor antigens. Three of the tested methods: allele-specific PCR, restriction fragment length polymorphism and real-time PCR with TaqMan probes demonstrated 100% reliability when compared to Sanger sequencing for all of the studied polymorphisms. High resolution melting analysis was unsuitable for genotyping of one of the tested minor antigens (HA-1 as it has linked synonymous polymorphism. Obtained data could be used to select the strategy for large-scale clinical genotyping.

  3. Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk.

    Science.gov (United States)

    Martín, Maria Cruz; del Rio, Beatriz; Martínez, Noelia; Magadán, Alfonso H; Alvarez, Miguel A

    2008-12-01

    One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.

  4. Molecular detection of oomycetes species in water courses

    Directory of Open Access Journals (Sweden)

    Oszako Tomasz

    2016-12-01

    Full Text Available In Poland, about 20% of forest nurseries use irrigation water coming from natural superficial reservoirs, presumed to be the first source of infection caused by harmful pathogens belonging to the Oomycota class, especially Phytophthora genus and Pythium genus. The forest nursery is the only place where forest managers can react before pathogens leave it with asymptomatic plants or soil attached to their roots. The aim of this research was detection and identification phytopathogens in water samples. In order to recognise genus Phytophthora or Pythium in water collected from 33 places in five different forest districts in Poland, two DNA-based approaches of identification were applied: (i the TaqMan probes, and (ii sequencing of the ITS6/4 region.

  5. Preliminary report for analysis of genome wide mutations from four ciprofloxacin resistant B. anthracis Sterne isolates generated by Illumina, 454 sequencing and microarrays for DHS

    Energy Technology Data Exchange (ETDEWEB)

    Jaing, Crystal [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Vergez, Lisa [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Hinckley, Aubree [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Thissen, James [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Gardner, Shea [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); McLoughlin, Kevin [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Jackson, Paul [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Ellingson, Sally [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Hauser, Loren [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Brettin, Tom [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Fofanov, Viacheslav [Eureka Genomics, Hercules, CA (United States); Koshinsky, Heather [Eureka Genomics, Hercules, CA (United States); Fofanov, Yuriy [Univ. of Houston, TX (United States)

    2011-06-21

    The objective of this project is to provide DHS a comprehensive evaluation of the current genomic technologies including genotyping, Taqman PCR, multiple locus variable tandem repeat analysis (MLVA), microarray and high-throughput DNA sequencing in the analysis of biothreat agents from complex environmental samples. As the result of a different DHS project, we have selected for and isolated a large number of ciprofloxacin resistant B. anthracis Sterne isolates. These isolates vary in the concentrations of ciprofloxacin that they can tolerate, suggesting multiple mutations in the samples. In collaboration with University of Houston, Eureka Genomics and Oak Ridge National Laboratory, we analyzed the ciprofloxacin resistant B. anthracis Sterne isolates by microarray hybridization, Illumina and Roche 454 sequencing to understand the error rates and sensitivity of the different methods. The report provides an assessment of the results and a complete set of all protocols used and all data generated along with information to interpret the protocols and data sets.

  6. Real-Time PCR Detection of Dogwood Anthracnose Fungus in Historical Herbarium Specimens from Asia.

    Science.gov (United States)

    Miller, Stephen; Masuya, Hayato; Zhang, Jian; Walsh, Emily; Zhang, Ning

    2016-01-01

    Cornus species (dogwoods) are popular ornamental trees and important understory plants in natural forests of northern hemisphere. Dogwood anthracnose, one of the major diseases affecting the native North American Cornus species, such as C. florida, is caused by the fungal pathogen Discula destructiva. The origin of this fungus is not known, but it is hypothesized that it was imported to North America with its host plants from Asia. In this study, a TaqMan real-time PCR assay was used to detect D. destructiva in dried herbarium and fresh Cornus samples. Several herbarium specimens from Japan and China were detected positive for D. destructiva, some of which were collected before the first report of the dogwood anthracnose in North America. Our findings further support that D. destructiva was introduced to North America from Asia where the fungus likely does not cause severe disease.

  7. Quantification of massively parallel sequencing libraries - a comparative study of eight methods

    DEFF Research Database (Denmark)

    Hussing, Christian; Kampmann, Marie-Louise; Mogensen, Helle Smidt

    2018-01-01

    Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification...... estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time......-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out....

  8. Age- and Tumor Subtype-Specific Breast Cancer Risk Estimates for CHEK2*1100delC Carriers

    DEFF Research Database (Denmark)

    Schmidt, Marjanka K; Hogervorst, Frans; van Hien, Richard R

    2016-01-01

    PURPOSE: CHEK2*1100delC is a well-established breast cancer risk variant that is most prevalent in European populations; however, there are limited data on risk of breast cancer by age and tumor subtype, which limits its usefulness in breast cancer risk prediction. We aimed to generate tumor...... subtype- and age-specific risk estimates by using data from the Breast Cancer Association Consortium, including 44,777 patients with breast cancer and 42,997 controls from 33 studies genotyped for CHEK2*1100delC. PATIENTS AND METHODS: CHEK2*1100delC genotyping was mostly done by a custom Taqman assay....... Breast cancer odds ratios (ORs) for CHEK2*1100delC carriers versus noncarriers were estimated by using logistic regression and adjusted for study (categorical) and age. Main analyses included patients with invasive breast cancer from population- and hospital-based studies. RESULTS: Proportions...

  9. E112D polymorphism in the prolylcarboxypeptidase gene is associated with blood pressure response to benazepril in Chinese hypertensive patients.

    Science.gov (United States)

    Zhang, Yan; Hong, Xiu-mei; Xing, Hou-xun; Li, Jian-ping; Huo, Yong; Xu, Xi-ping

    2009-10-20

    Marked interindividual variation exists in blood pressure response to benazepril, which is considered to have genetic basis. Our objectives were to evaluate whether the E112D polymorphism in the prolylcarboxypeptidase (PRCP) gene has impact on blood pressure response to benazepril. Hypertensive patients from Huoqiu County and Yuexi County of Anhui Province received daily treatment with an oral dosage of 10 mg benazepril for 15 days. Genotypes of the E112D polymorphism in the PRCP gene were determined by TaqMan SNP genotyping assay. Multivariate linear and Logistic regressions using generalized estimating equation model were performed in a total of 1092 patients to evaluate the association of PRCP genotypes and blood pressure response to benazepril. Patients carrying ED or DD genotype had a less systolic blood pressure reduction (adjusted beta = -3.7 + or - 1.1, P benazepril treatment in hypertensive patients of Anhui Province, China.

  10. Novel reverse transcription loop-mediated isothermal amplification for rapid detection of foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Dukes, J.P.; King, D.P.; Alexandersen, Søren

    2006-01-01

    Speed is paramount in the diagnosis of foot-and-mouth disease (FMD) and simplicity is required if a test is to be deployed in the field. The development of a one-step, reverse transcription loop-mediated amplification (RT-LAMP) assay enables FMD virus (FMDV) to be detected in under an hour...... in a single tube without thermal cycling. A fragment of the 3D RNA polymerase gene of the virus is amplified at 65 degrees C in the presence of a primer mixture and both reverse transcriptase and Bst DNA polymerase. Compared with real-time RT-PCR, RT-LAMP was consistently faster, and ten copies of FMDV...... vesicular diseases and from that of genetically related picornaviruses. Diagnostic sensitivity was validated by the amplification of reference FMDV strains and archival material from field cases of FMD. In comparison with the performance of the established diagnostic TaqMan (R) assay, RT-LAMP appears...

  11. High-resolution melting analysis using unlabeled probe and amplicon scanning simultaneously detects several lactase persistence variants

    DEFF Research Database (Denmark)

    Janukonyté, Jurgita; Vestergaard, Else M; Ladefoged, Søren A

    2010-01-01

    Lactase persistence and thereby tolerance to lactose is a common trait in people of Northern European descent. It is linked to the LCT -13910C>T variant located in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase (LCT) gene. In people of African and Middle Eastern descent, lactase...... persistence can be associated with other variants nearby the -13910C>T variant, limiting the use of the -13910C>T-based SNP analysis, e.g. TaqMan assays for the diagnosis of lactose intolerance. Using high-resolution melting analysis, we identified five samples that were heterozygous for the -13915T>G variant...... the -13910C>T and -13915T>G variants in addition to rarer variants surrounding the -13910 site. This new method may contribute to improve the diagnostic performance of the genetic analysis for lactose intolerance....

  12. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

    DEFF Research Database (Denmark)

    Wang, Chong; Robles, Francisco; Ramirez, Saul

    2016-01-01

    Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real...... published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10......-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial...

  13. Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

    DEFF Research Database (Denmark)

    Hakhverdyan, Mikhayil; Hägglund, Sara; Larsen, Lars Erik

    2005-01-01

    understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format......, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR......, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can...

  14. Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS® Ampliprep/COBAS®TaqMan HBV test v2.0.

    Science.gov (United States)

    Salpini, Romina; Piermatteo, Lorenzo; Gill, Upkar; Battisti, Arianna; Stazi, Francesca; Guenci, Tania; Giannella, Sara; Serafini, Valentina; Kennedy, Patrick T F; Perno, Carlo Federico; Svicher, Valentina; Ciotti, Marco

    2017-08-01

    Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBAS ® Ampliprep/COBAS ® TaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R 2  = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R 2  = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBAS ® Ampliprep/COBAS ® TaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.

  15. [Quantitative fluorogenic real-time PCR assay for respiratory syncytial virus detection].

    Science.gov (United States)

    Zhang, Qi-wei; You, Shang-you; Sun, Ji-min; Wu, Qi; Yu, Chun-hua; Zhang, Chu-yu

    2005-07-01

    To Establish a rapid and objective quantitative fluorogenic real-time PCR assay for early detection of human respiratory syncytial virus (hRSV). Two pairs of primers and one TaqMan Fluorogenic probe that are specific for the recognition of the most conservative N gene of hRSV for virus detection with LighCycler PCR in 93 nasopharyngeal secretion specimens collected from infants and young children. The assay was compared with virus isolation, routine PCR, nested PCR, and enzyme-linked immunosorbent assay (ELISA). This TaqMan assay had a sensitivity of 1 x 10(2) cDNA copies/microl with a dynamic range between 1 x 10(2) and 1 x 10(7) cDNA copies/microl, which was the same as that of nested PCR, but 10 times more sensitive than routine PCR. The specificity of the assay was evaluated by comparing hRSV with polivirus type 1, coxsackie virus type 2, influenza A, influenza B and adenovirus type 7. A PCR product of the expected size (195 bp) was produced and fluorescence signal detected for hRSV, but not for any of the other viruses. The results in LightCycler and Rotor-Gene instrument were consistent. Forty-four specimens (43.9%) were hRSV-positive with this assay and 4 (4/93,4.3%) were hRSV-positive with ELISA, showing rather low correlation between the two methods. No visible relation was found between the concentration of hRSV RNA and severity of the disease. This assay is rapid, sensitive, specific and quantitative, and has the potential of wide application for early diagnosis of hRSV infection and evaluation of the therapeutic effect.

  16. A Genome Wide Study of Copy Number Variation Associated with Nasopharyngeal Carcinoma in Malaysian Chinese Identifies CNVs at 11q14.3 and 6p21.3 as Candidate Loci

    Science.gov (United States)

    Low, Joyce Siew Yong; Chin, Yoon Ming; Mushiroda, Taisei; Kubo, Michiaki; Govindasamy, Gopala Krishnan; Pua, Kin Choo; Yap, Yoke Yeow; Yap, Lee Fah; Subramaniam, Selva Kumar; Ong, Cheng Ai; Tan, Tee Yong; Khoo, Alan Soo Beng; Ng, Ching Ching

    2016-01-01

    Background Nasopharyngeal carcinoma (NPC) is a neoplasm of the epithelial lining of the nasopharynx. Despite various reports linking genomic variants to NPC predisposition, very few reports were done on copy number variations (CNV). CNV is an inherent structural variation that has been found to be involved in cancer predisposition. Methods A discovery cohort of Malaysian Chinese descent (NPC patients, n = 140; Healthy controls, n = 256) were genotyped using Illumina® HumanOmniExpress BeadChip. PennCNV and cnvPartition calling algorithms were applied for CNV calling. Taqman CNV assays and digital PCR were used to validate CNV calls and replicate candidate copy number variant region (CNVR) associations in a follow-up Malaysian Chinese (NPC cases, n = 465; and Healthy controls, n = 677) and Malay cohort (NPC cases, n = 114; Healthy controls, n = 124). Results Six putative CNVRs overlapping GRM5, MICA/HCP5/HCG26, LILRB3/LILRA6, DPY19L2, RNase3/RNase2 and GOLPH3 genes were jointly identified by PennCNV and cnvPartition. CNVs overlapping GRM5 and MICA/HCP5/HCG26 were subjected to further validation by Taqman CNV assays and digital PCR. Combined analysis in Malaysian Chinese cohort revealed a strong association at CNVR on chromosome 11q14.3 (Pcombined = 1.54x10-5; odds ratio (OR) = 7.27; 95% CI = 2.96–17.88) overlapping GRM5 and a suggestive association at CNVR on chromosome 6p21.3 (Pcombined = 1.29x10-3; OR = 4.21; 95% CI = 1.75–10.11) overlapping MICA/HCP5/HCG26 genes. Conclusion Our results demonstrated the association of CNVs towards NPC susceptibility, implicating a possible role of CNVs in NPC development. PMID:26730743

  17. A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking

    Directory of Open Access Journals (Sweden)

    Rosen David

    2008-10-01

    Full Text Available Abstract Background Single nucleotide polymorphism (SNP genotyping provides the means to develop a practical, rapid, inexpensive assay that will uniquely identify any Plasmodium falciparum parasite using a small amount of DNA. Such an assay could be used to distinguish recrudescence from re-infection in drug trials, to monitor the frequency and distribution of specific parasites in a patient population undergoing drug treatment or vaccine challenge, or for tracking samples and determining purity of isolates in the laboratory during culture adaptation and sub-cloning, as well as routine passage. Methods A panel of twenty-four SNP markers has been identified that exhibit a high minor allele frequency (average MAF > 35%, for which robust TaqMan genotyping assays were constructed. All SNPs were identified through whole genome sequencing and MAF was estimated through Affymetrix array-based genotyping of a worldwide collection of parasites. These assays create a "molecular barcode" to uniquely identify a parasite genome. Results Using 24 such markers no two parasites known to be of independent origin have yet been found to have the same allele signature. The TaqMan genotyping assays can be performed on a variety of samples including cultured parasites, frozen whole blood, or whole blood spotted onto filter paper with a success rate > 99%. Less than 5 ng of parasite DNA is needed to complete a panel of 24 markers. The ability of this SNP panel to detect and identify parasites was compared to the standard molecular methods, MSP-1 and MSP-2 typing. Conclusion This work provides a facile field-deployable genotyping tool that can be used without special skills with standard lab equipment, and at reasonable cost that will unambiguously identify and track P. falciparum parasites both from patient samples and in the laboratory.

  18. A Genome Wide Study of Copy Number Variation Associated with Nasopharyngeal Carcinoma in Malaysian Chinese Identifies CNVs at 11q14.3 and 6p21.3 as Candidate Loci.

    Directory of Open Access Journals (Sweden)

    Joyce Siew Yong Low

    Full Text Available Nasopharyngeal carcinoma (NPC is a neoplasm of the epithelial lining of the nasopharynx. Despite various reports linking genomic variants to NPC predisposition, very few reports were done on copy number variations (CNV. CNV is an inherent structural variation that has been found to be involved in cancer predisposition.A discovery cohort of Malaysian Chinese descent (NPC patients, n = 140; Healthy controls, n = 256 were genotyped using Illumina® HumanOmniExpress BeadChip. PennCNV and cnvPartition calling algorithms were applied for CNV calling. Taqman CNV assays and digital PCR were used to validate CNV calls and replicate candidate copy number variant region (CNVR associations in a follow-up Malaysian Chinese (NPC cases, n = 465; and Healthy controls, n = 677 and Malay cohort (NPC cases, n = 114; Healthy controls, n = 124.Six putative CNVRs overlapping GRM5, MICA/HCP5/HCG26, LILRB3/LILRA6, DPY19L2, RNase3/RNase2 and GOLPH3 genes were jointly identified by PennCNV and cnvPartition. CNVs overlapping GRM5 and MICA/HCP5/HCG26 were subjected to further validation by Taqman CNV assays and digital PCR. Combined analysis in Malaysian Chinese cohort revealed a strong association at CNVR on chromosome 11q14.3 (Pcombined = 1.54x10-5; odds ratio (OR = 7.27; 95% CI = 2.96-17.88 overlapping GRM5 and a suggestive association at CNVR on chromosome 6p21.3 (Pcombined = 1.29x10-3; OR = 4.21; 95% CI = 1.75-10.11 overlapping MICA/HCP5/HCG26 genes.Our results demonstrated the association of CNVs towards NPC susceptibility, implicating a possible role of CNVs in NPC development.

  19. A 7 gene expression score predicts for radiation response in cancer cervix

    International Nuclear Information System (INIS)

    Rajkumar, Thangarajan; Vijayalakshmi, Neelakantan; Sabitha, Kesavan; Shirley, Sundersingh; Selvaluxmy, Ganesharaja; Bose, Mayil Vahanan; Nambaru, Lavanya

    2009-01-01

    Cervical cancer is the most common cancer among Indian women. The current recommendations are to treat the stage IIB, IIIA, IIIB and IVA with radical radiotherapy and weekly cisplatin based chemotherapy. However, Radiotherapy alone can help cure more than 60% of stage IIB and up to 40% of stage IIIB patients. Archival RNA samples from 15 patients who had achieved complete remission and stayed disease free for more than 36 months (No Evidence of Disease or NED group) and 10 patients who had failed radical radiotherapy (Failed group) were included in the study. The RNA were amplified, labelled and hybridized to Stanford microarray chips and analyzed using BRB Array Tools software and Significance Analysis of Microarray (SAM) analysis. 20 genes were selected for further validation using Relative Quantitation (RQ) Taqman assay in a Taqman Low-Density Array (TLDA) format. The RQ value was calculated, using each of the NED sample once as a calibrator. A scoring system was developed based on the RQ value for the genes. Using a seven gene based scoring system, it was possible to distinguish between the tumours which were likely to respond to the radiotherapy and those likely to fail. The mean score ± 2 SE (standard error of mean) was used and at a cut-off score of greater than 5.60, the sensitivity, specificity, Positive predictive value (PPV) and Negative predictive value (NPV) were 0.64, 1.0, 1.0, 0.67, respectively, for the low risk group. We have identified a 7 gene signature which could help identify patients with cervical cancer who can be treated with radiotherapy alone. However, this needs to be validated in a larger patient population

  20. Bone marker gene expression in calvarial bones: different bone microenvironments.

    Science.gov (United States)

    Al-Amer, Osama

    2017-12-01

    In calvarial mice, mesenchymal stem cells (MSCs) differentiate into osteoprogenitor cells and then differentiate into osteoblasts that differentiate into osteocytes, which become embedded within the bone matrix. In this case, the cells participating in bone formation include MSCs, osteoprogenitor cells, osteoblasts and osteocytes. The calvariae of C57BL/KaLwRijHsD mice consist of the following five bones: two frontal bones, two parietal bones and one interparietal bone. This study aimed to analyse some bone marker genes and bone related genes to determine whether these calvarial bones have different bone microenvironments. C57BL/KaLwRijHsD calvariae were carefully excised from five male mice that were 4-6 weeks of age. Frontal, parietal, and interparietal bones were dissected to determine the bone microenvironment in calvariae. Haematoxylin and eosin staining was used to determine the morphology of different calvarial bones under microscopy. TaqMan was used to analyse the relative expression of Runx2, OC, OSX, RANK, RANKL, OPG, N-cadherin, E-cadherin, FGF2 and FGFR1 genes in different parts of the calvariae. Histological analysis demonstrated different bone marrow (BM) areas between the different parts of the calvariae. The data show that parietal bones have the smallest BM area compared to frontal and interparietal bones. TaqMan data show a significant increase in the expression level of Runx2, OC, OSX, RANKL, OPG, FGF2 and FGFR1 genes in the parietal bones compared with the frontal and interparietal bones of calvariae. This study provides evidence that different calvarial bones, frontal, parietal and interparietal, contain different bone microenvironments.

  1. Expectoration of Flaviviruses during sugar feeding by mosquitoes (Diptera: Culicidae).

    Science.gov (United States)

    van den Hurk, Andrew F; Johnson, Petrina H; Hall-Mendelin, Sonja; Northill, Judy A; Simmons, Russell J; Jansen, Cassie C; Frances, Stephen P; Smith, Greg A; Ritchie, Scott A

    2007-09-01

    Biological transmission of arboviruses to a vertebrate host occurs when virions are expelled along with saliva during blood feeding by a hematophagous arthropod. We undertook experiments to determine whether mosquitoes expectorate flaviviruses in their saliva while sugar feeding. Batches of Culex annulirostris Skuse and Culex gelidus Theobald (Diptera: Culicidae) were orally infected with Japanese encephalitis (family Flaviviridae, genus Flavivirus, JEV), Kunjin (family Flaviviridae, genus Flavivirus, KUNV; a subtype of West Nile virus), and Murray Valley encephalitis (family Flaviviridae, genus Flavivirus, MVEV) viruses. After a 7-d extrinsic incubation, these mosquitoes were offered sucrose meals via cotton pledgets, which were removed daily and processed for viral RNA by using real-time TaqMan reverse transcriptase-polymerase chain reaction (RT-PCR) assays. JEV, MVEV, and KUNV RNA was detected in all pledgets removed from batches of Cx. gelidus on days 7-14 postexposure. In contrast, detection rates were variable for Cx. annulirostris, with KUNV detected in 0.3 M sucrose pledgets on all days postexposure, and JEV and MVEV detected on 57 and 50% of days postexposure, respectively. Higher concentrations of sucrose in the pledget did not increase virus detection rates. When individual JEV-infected Cx. gelidus were exposed to the sucrose pledget, 73% of mosquitoes expectorated virus with titers that were detectable by TaqMan RT-PCR. These results clearly show that flaviviruses are expectorated by infected mosquitoes during the process of sugar feeding on artificial pledgets. Potential applications of the method for arboviral bioassays and field surveillance are discussed.

  2. Sequence-specific inhibition of microRNA-130a gene by CRISPR/Cas9 system in breast cancer cell line

    Science.gov (United States)

    Ainina Abdollah, Nur; Das Kumitaa, Theva; Yusof Narazah, Mohd; Razak, Siti Razila Abdul

    2017-05-01

    MicroRNAs (miRNAs) are short stranded noncoding RNA that play important roles in apoptosis, cell survival, development and cell proliferation. However, gene expression control via small regulatory RNA, particularly miRNA in breast cancer is still less explored. Therefore, this project aims to develop an approach to target microRNA-130a using the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system in MCF7, breast cancer cell line. The 20 bp sequences target at stem loop, 3ʹ and 5ʹ end of miR130a were cloned into pSpCas9(BB)-2A-GFP (PX458) plasmid, and the positive clones were confirmed by sequencing. A total of 5 μg of PX458-miR130a was transfected to MCF7 using Lipofectamine® 3000 according to manufacturer’s protocol. The transfected cells were maintained in the incubator at 37 °C under humidified 5% CO2. After 48 hours, cells were harvested and total RNA was extracted using miRNeasy Mini Kit (Qiagen). cDNAs were synthesised specific to miR-130a using TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). Then, qRT-PCR was carried out using TaqMan Universal Master Mix (Applied Biosystems) to quantify the knockdown level of mature miRNAs in the cells. Result showed that miR-130a-5p was significantly downregulated in MCF7 cell line. However, no significant changes were observed for sequences targeting miR-130a-3p and stem loop. Thus, this study showed that the expression of miR-130a-5p was successfully down-regulated using CRISPR silencing system. This technique may be useful to manipulate the level of miRNA in various cell types to answer clinical questions at the molecular level.

  3. Real-time PCR detection of Paenibacillus spp. in raw milk to predict shelf life performance of pasteurized fluid milk products.

    Science.gov (United States)

    Ranieri, Matthew L; Ivy, Reid A; Mitchell, W Robert; Call, Emma; Masiello, Stephanie N; Wiedmann, Martin; Boor, Kathryn J

    2012-08-01

    Psychrotolerant sporeformers, specifically Paenibacillus spp., are important spoilage bacteria for pasteurized, refrigerated foods such as fluid milk. While Paenibacillus spp. have been isolated from farm environments, raw milk, processing plant environments, and pasteurized fluid milk, no information on the number of Paenibacillus spp. that need to be present in raw milk to cause pasteurized milk spoilage was available. A real-time PCR assay targeting the 16S rRNA gene was designed to detect Paenibacillus spp. in fluid milk and to discriminate between Paenibacillus and other closely related spore-forming bacteria. Specificity was confirmed using 16 Paenibacillus and 17 Bacillus isolates. All 16 Paenibacillus isolates were detected with a mean cycle threshold (C(T)) of 19.14 ± 0.54. While 14/17 Bacillus isolates showed no signal (C(T) > 40), 3 Bacillus isolates showed very weak positive signals (C(T) = 38.66 ± 0.65). The assay provided a detection limit of approximately 3.25 × 10(1) CFU/ml using total genomic DNA extracted from raw milk samples inoculated with Paenibacillus. Application of the TaqMan PCR to colony lysates obtained from heat-treated and enriched raw milk provided fast and accurate detection of Paenibacillus. Heat-treated milk samples where Paenibacillus (≥1 CFU/ml) was detected by this colony TaqMan PCR showed high bacterial counts (>4.30 log CFU/ml) after refrigerated storage (6°C) for 21 days. We thus developed a tool for rapid detection of Paenibacillus that has the potential to identify raw milk with microbial spoilage potential as a pasteurized product.

  4. Rapid and sensitive approach to simultaneous detection of genomes of hepatitis A, B, C, D and E viruses.

    Science.gov (United States)

    Kodani, Maja; Mixson-Hayden, Tonya; Drobeniuc, Jan; Kamili, Saleem

    2014-10-01

    Five viruses have been etiologically associated with viral hepatitis. Nucleic acid testing (NAT) remains the gold standard for diagnosis of viremic stages of infection. NAT methodologies have been developed for all hepatitis viruses; however, a NAT-based assay that can simultaneously detect all five viruses is not available. We designed TaqMan card-based assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. The performances of individual assays were evaluated on TaqMan Array Cards (TAC) for detecting five viral genomes simultaneously. Sensitivity and specificity were determined by testing 329 NAT-tested clinical specimens. All NAT-positive samples for HCV (n = 32), HDV (n = 28) and HEV (n = 14) were also found positive in TAC (sensitivity, 100%). Forty-three of 46 HAV-NAT positive samples were also positive in TAC (sensitivity, 94%), while 36 of 39 HBV-NAT positive samples were positive (sensitivity, 92%). No false-positives were detected for HBV (n = 32), HCV (n = 36), HDV (n = 30), and HEV (n = 31) NAT-negative samples (specificity 100%), while 38 of 41 HAV-NAT negative samples were negative by TAC (specificity 93%). TAC assay was concordant with corresponding individual NATs for hepatitis A-E viral genomes and can be used for their detection simultaneously. The TAC assay has potential for use in hepatitis surveillance, for screening of donor specimens and in outbreak situations. Wider availability of TAC-ready assays may allow for customized assays, for improving acute jaundice surveillance and for other purposes for which there is need to identify multiple pathogens rapidly. Published by Elsevier B.V.

  5. Usefulness of in-house PCR methods for hepatitis B virus DNA detection.

    Science.gov (United States)

    Portilho, Moyra Machado; Baptista, Marcia Leite; da Silva, Messias; de Sousa, Paulo Sérgio Fonseca; Lewis-Ximenez, Lia Laura; Lampe, Elisabeth; Villar, Livia Melo

    2015-10-01

    The aim of the present study was to evaluate the performance of three in-house PCR techniques for HBV DNA detection and compare it with commercial quantitative methods to evaluate the usefulness of in-house methods for HBV diagnosis. Three panels of HBsAg reactive sera samples were evaluated: (i) 50 samples were examined using three methods for in-house qualitative PCR and the Cobas Amplicor HBV Monitor Assay; (ii) 87 samples were assayed using in-house semi-nested PCR and the Cobas TaqMan HBV test; (iii) 11 serial samples obtained from 2 HBV-infected individuals were assayed using the Cobas Amplicor HBV test and semi-nested PCR. In panel I, HBV DNA was detected in 44 samples using the Cobas Amplicor HBV test, 42 samples using semi-nested PCR (90% concordance with Cobas Amplicor), 22 samples using PCR for the core gene (63.6% concordance) and 29 samples using single-round PCR for the pre-S/S gene (75% concordance). In panel II, HBV DNA was quantified in 78 of the 87 HBsAg reactive samples using Cobas TaqMan but 52 samples using semi-nested PCR (67.8% concordance). HBV DNA was detected in serial samples until the 17th and 26th week after first donation using in-house semi-nested PCR and the Cobas Amplicor HBV test, respectively. In-house semi-nested PCR presented adequate concordance with commercial methods as an alternative method for HBV molecular diagnosis in low-resource settings. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2014-08-01

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

  7. A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3).

    Science.gov (United States)

    Glenney, Gavin W; Barbash, Patricia A; Coll, John A

    2016-03-01

    Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

  8. Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting.

    Directory of Open Access Journals (Sweden)

    Steven Y C Tong

    Full Text Available We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR, high-resolution melting (HRM assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T was 32.98 would have an H275Y assay C(T>30. Analysis of the TaqMan C(T values for 609 consecutive clinical samples predicted that 207 (34% of the samples would result in an HRM assay C(T>30 and therefore not be amenable to the HRM assay.The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m against C(T. Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.

  9. Detection, quantification and genotyping of Herpes Simplex Virus in cervicovaginal secretions by real-time PCR: a cross sectional survey

    Directory of Open Access Journals (Sweden)

    Natividad-Sancho Angels

    2005-08-01

    Full Text Available Abstract Background Herpes Simplex Virus (HSV Genital Ulcer Disease (GUD is an important public health problem, whose interaction with HIV results in mutually enhancing epidemics. Conventional methods for detecting HSV tend to be slow and insensitive. We designed a rapid PCR-based assay to quantify and type HSV in cervicovaginal lavage (CVL fluid of subjects attending a Genito-Urinary Medicine (GUM clinic. Vaginal swabs, CVL fluid and venous blood were collected. Quantitative detection of HSV was conducted using real time PCR with HSV specific primers and SYBR Green I. Fluorogenic TaqMan Minor Groove Binder (MGB probes designed around a single base mismatch in the HSV DNA polymerase I gene were used to type HSV in a separate reaction. The Kalon test was used to detect anti-HSV-2 IgG antibodies in serum. Testing for HIV, other Sexually Transmitted Infections (STI and related infections was based on standard clinical and laboratory methods. Results Seventy consecutive GUM clinic attendees were studied. Twenty-seven subjects (39% had detectable HSV DNA in CVL fluid; HSV-2 alone was detected in 19 (70% subjects, HSV-1 alone was detected in 4 (15% subjects and both HSV types were detected in 4 (15% subjects. Eleven out of 27 subjects (41% with anti-HSV-2 IgG had detectable HSV-2 DNA in CVL fluid. Seven subjects (10% were HIV-positive. Three of seven (43% HIV-infected subjects and two of five subjects with GUD (40% were secreting HSV-2. None of the subjects in whom HSV-1 was detected had GUD. Conclusion Quantitative real-time PCR and Taqman MGB probes specific for HSV-1 or -2 were used to develop an assay for quantification and typing of HSV. The majority of subjects in which HSV was detected had low levels of CVL fluid HSV, with no detectable HSV-2 antibodies and were asymptomatic.

  10. New miRNA labeling method for bead-based quantification

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    Lanfranchi Gerolamo

    2010-06-01

    Full Text Available Abstract Background microRNAs (miRNAs are small single-stranded non-coding RNAs that act as crucial regulators of gene expression. Different methods have been developed for miRNA expression profiling in order to better understand gene regulation in normal and pathological conditions. miRNAs expression values obtained from large scale methodologies such as microarrays still need a validation step with alternative technologies. Results Here we have applied with an innovative approach, the Luminex® xMAP™ technology validate expression data of differentially expressed miRNAs obtained from high throughput arrays. We have developed a novel labeling system of small RNA molecules (below 200 nt, optimizing the sensitive cloning method for miRNAs, termed miRNA amplification profiling (mRAP. The Luminex expression patterns of three miRNAs (miR-23a, miR-27a and miR-199a in seven different cell lines have been validated by TaqMan miRNA assay. In all cases, bead-based meas were confirmed by the data obtained by TaqMan and microarray technologies. Conclusions We demonstrate that the measure of individual miRNA by the bead-based method is feasible, high speed, sensitive and low cost. The Luminex® xMAP™ technology also provides flexibility, since the central reaction can be scaled up with additional miRNA capturing beads, allowing validation of many differentially expressed miRNAs obtained from microarrays in a single experiment. We propose this technology as an alternative method to qRT-PCR for validating miRNAs expression data obtained with high-throughput technologies.

  11. Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts.

    Science.gov (United States)

    Samuel, Stylianos; Papayiannis, Lambros C; Leroch, Michaela; Veloukas, Thomas; Hahn, Matthias; Karaoglanidis, George S

    2011-08-01

    Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005-2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real-time TaqMan PCR assay developed in the present study. QoI-resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse-grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI-resistant and QoI-sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. The results of the study suggest that a high risk for selection of QoI-resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real-time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre- and post-amplification manipulations, and can be used for rapid screening and quantification of QoI resistance. Copyright © 2011 Society of Chemical Industry.

  12. Recurrent high level parvovirus B19/genotype 2 viremia in a renal transplant recipient analyzed by real-time PCR for simultaneous detection of genotypes 1 to 3.

    Science.gov (United States)

    Liefeldt, Lutz; Plentz, Annelie; Klempa, Boris; Kershaw, Olivia; Endres, Anne-Sophie; Raab, Ulla; Neumayer, Hans-H; Meisel, Helga; Modrow, Susanne

    2005-01-01

    Organ transplant recipients infected with parvovirus B19 frequently develop persistent viremia associated with chronic anemia and pure red cell aplasia. In this study, a male renal transplant recipient who had been infected with parvovirus B19/genotype 2 after renal transplantation at the age of 34 years is described. The patient was repeatedly treated with high dose intravenous immunoglobulin (IVIG) that resulted in the resolvement of symptoms but not in virus eradication. During an observation period of 33 months after transplantation three phases associated with high parvovirus B19 viremia were observed. Both the first and the second viremic phases were combined with severe anemia. Parvovirus B19 specific IgM-antibodies were initially detected at the beginning of the second phase in continually rising concentrations. Initially eradication of the virus by immunoglobulin therapy was reported after the first viremic phase [Liefeldt et al. (2002): Nephrol Dial Transplant 17:1840-1842]. Retrospectively this statement has to be corrected. It was based on the use of a qualitative PCR assay specific for parvovirus B19 genotype 1 associated with reduced sensitivity for detection of genotype 2. After sequence analysis of the viral DNA and adjustment of a real-time PCR assay (TaqMan) for quantitative detection of all three B19 virus genotypes analysis of consecutive serum samples allowed the demonstration of long lasting phases with reduced viral loads following IVIG-treatment. These results demonstrate that IVIG treatment of parvovirus B19-triggered anemia in transplant recipients offers an opportunity to resolve symptoms, but does not guarantee eradication of the virus. Since reactivation of parvovirus B19 infection can result in high virus load associated with the recurrence of symptoms repeated screening for viral DNA is recommended using the TaqMan system established for quantitative detection of all three genotypes of parvovirus B19. Copyright 2005 Wiley-Liss, Inc.

  13. Development of a highly sensitive real-time nested RT-PCR assay in a single closed tube for detection of enterovirus 71 in hand, foot, and mouth disease.

    Science.gov (United States)

    Niu, Peihua; Qi, Shunxiang; Yu, Benzhang; Zhang, Chen; Wang, Ji; Li, Qi; Ma, Xuejun

    2016-11-01

    Enterovirus 71 (EV71) is one of the major causative agents of outbreaks of hand, foot, and mouth disease (HFMD). A commercial TaqMan probe-based real-time PCR assay has been widely used for the differential detection of EV71 despite its relatively high cost and failure to detect samples with a low viral load (Ct value > 35). In this study, a highly sensitive real-time nested RT-PCR (RTN RT-PCR) assay in a single closed tube for detection of EV71 in HFMD was developed. The sensitivity and specificity of this assay were evaluated using a reference EV71 stock and a panel of controls consisting of coxsackievirus A16 (CVA16) and common respiratory viruses, respectively. The clinical performance of this assay was evaluated and compared with those of a commercial TaqMan probe-based real-time PCR (qRT-PCR) assay and a traditional two-step nested RT-PCR assay. The limit of detection for the RTN RT-PCR assay was 0.01 TCID50/ml, with a Ct value of 38.3, which was the same as that of the traditional two-step nested RT-PCR assay and approximately tenfold lower than that of the qRT-PCR assay. When testing the reference strain EV71, this assay showed favorable detection reproducibility and no obvious cross-reactivity. The testing results of 100 clinical throat swabs from HFMD-suspected patients revealed that 41 samples were positive for EV71 by both RTN RT-PCR and traditional two-step nested RT-PCR assays, whereas only 29 were EV71 positive by qRT-PCR assay.

  14. Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.

    Science.gov (United States)

    Zarrinfar, Hossein; Makimura, Koichi; Satoh, Kazuo; Khodadadi, Hossein; Mirhendi, Hossein

    2013-05-01

    Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group. © 2013 Wiley Periodicals, Inc.

  15. Canonical Single Nucleotide Polymorphisms (SNPs for High-Resolution Subtyping of Shiga-Toxin Producing Escherichia coli (STEC O157:H7.

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    Sean M Griffing

    Full Text Available The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing Escherichia coli (STEC O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide

  16. Transcript and protein analysis reveals better survival skills of monocyte-derived dendritic cells compared to monocytes during oxidative stress.

    Directory of Open Access Journals (Sweden)

    Ilse Van Brussel

    Full Text Available BACKGROUND: Dendritic cells (DCs, professional antigen-presenting cells with the unique ability to initiate primary T-cell responses, are present in atherosclerotic lesions where they are exposed to oxidative stress that generates cytotoxic reactive oxygen species (ROS. A large body of evidence indicates that cell death is a major modulating factor of atherogenesis. We examined antioxidant defence systems of human monocyte-derived (moDCs and monocytes in response to oxidative stress. METHODS: Oxidative stress was induced by addition of tertiary-butylhydroperoxide (tert-BHP, 30 min. Cellular responses were evaluated using flow cytometry and confocal live cell imaging (both using 5-(and-6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate, CM-H(2DCFDA. Viability was assessed by the neutral red assay. Total RNA was extracted for a PCR profiler array. Five genes were selected for confirmation by Taqman gene expression assays, and by immunoblotting or immunohistochemistry for protein levels. RESULTS: Tert-BHP increased CM-H(2DCFDA fluorescence and caused cell death. Interestingly, all processes occurred more slowly in moDCs than in monocytes. The mRNA profiler array showed more than 2-fold differential expression of 32 oxidative stress-related genes in unstimulated moDCs, including peroxiredoxin-2 (PRDX2, an enzyme reducing hydrogen peroxide and lipid peroxides. PRDX2 upregulation was confirmed by Taqman assays, immunoblotting and immunohistochemistry. Silencing PRDX2 in moDCs by means of siRNA significantly increased CM-DCF fluorescence and cell death upon tert-BHP-stimulation. CONCLUSIONS: Our results indicate that moDCs exhibit higher intracellular antioxidant capacities, making them better equipped to resist oxidative stress than monocytes. Upregulation of PRDX2 is involved in the neutralization of ROS in moDCs. Taken together, this points to better survival skills of DCs in oxidative stress environments, such as atherosclerotic plaques.

  17. New views on strand asymmetry in insect mitochondrial genomes.

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    Shu-Jun Wei

    Full Text Available Strand asymmetry in nucleotide composition is a remarkable feature of animal mitochondrial genomes. Understanding the mutation processes that shape strand asymmetry is essential for comprehensive knowledge of genome evolution, demographical population history and accurate phylogenetic inference. Previous studies found that the relative contributions of different substitution types to strand asymmetry are associated with replication alone or both replication and transcription. However, the relative contributions of replication and transcription to strand asymmetry remain unclear. Here we conducted a broad survey of strand asymmetry across 120 insect mitochondrial genomes, with special reference to the correlation between the signs of skew values and replication orientation/gene direction. The results show that the sign of GC skew on entire mitochondrial genomes is reversed in all species of three distantly related families of insects, Philopteridae (Phthiraptera, Aleyrodidae (Hemiptera and Braconidae (Hymenoptera; the replication-related elements in the A+T-rich regions of these species are inverted, confirming that reversal of strand asymmetry (GC skew was caused by inversion of replication origin; and finally, the sign of GC skew value is associated with replication orientation but not with gene direction, while that of AT skew value varies with gene direction, replication and codon positions used in analyses. These findings show that deaminations during replication and other mutations contribute more than selection on amino acid sequences to strand compositions of G and C, and that the replication process has a stronger affect on A and T content than does transcription. Our results may contribute to genome-wide studies of replication and transcription mechanisms.

  18. Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

    Science.gov (United States)

    Anwar, R; Booth, A; Churchill, A J; Markham, A F

    1996-01-01

    The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

  19. miR-26b promotes granulosa cell apoptosis by targeting ATM during follicular atresia in porcine ovary.

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    Fei Lin

    Full Text Available More than 99% of ovarian follicles undergo atresia in mammals, but the mechanism of follicular atresia remains to be elucidated. In this study, we explored microRNA (miRNA regulation of follicular atresia in porcine ovary. A miRNA expression profile was constructed for healthy, early atretic, and progressively atretic follicles, and the differentially expressed miRNAs were selected and analyzed. We found that miR-26b, which was upregulated during follicular atresia, increased the number of DNA breaks and promoted granulosa cell apoptosis by targeting the ataxia telangiectasia mutated gene directly in vitro.

  20. The TAL1 complex targets the FBXW7 tumor suppressor by activating miR-223 in human T cell acute lymphoblastic leukemia

    OpenAIRE

    Mansour, Marc R.; Sanda, Takaomi; Lawton, Lee N.; Li, Xiaoyu; Kreslavsky, Taras; Novina, Carl D.; Brand, Marjorie; Gutierrez, Alejandro; Kelliher, Michelle A.; Jamieson, Catriona H.M.; von Boehmer, Harald; Young, Richard A.; Look, A. Thomas

    2013-01-01

    The oncogenic transcription factor TAL1/SCL is aberrantly expressed in 60% of cases of human T cell acute lymphoblastic leukemia (T-ALL) and initiates T-ALL in mouse models. By performing global microRNA (miRNA) expression profiling after depletion of TAL1, together with genome-wide analysis of TAL1 occupancy by chromatin immunoprecipitation coupled to massively parallel DNA sequencing, we identified the miRNA genes directly controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GAT...

  1. Real-time polymerase chain reaction-based approach for quantification of the pat gene in the T25 Zea mays event.

    Science.gov (United States)

    Weighardt, Florian; Barbati, Cristina; Paoletti, Claudia; Querci, Maddalena; Kay, Simon; De Beuckeleer, Marc; Van den Eede, Guy

    2004-01-01

    In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement

  2. Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli in Swine Feces Recovered in the National Animal Health Monitoring System's Swine 2000 Study

    Science.gov (United States)

    Fratamico, Pina M.; Bagi, Lori K.; Bush, Eric J.; Solow, Barbara T.

    2004-01-01

    A study was conducted to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in swine feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from 13 of the top 17 swine-producing states were tested for the presence of STEC. After enrichment of swine fecal samples in tryptic soy broth, the samples were tested for the presence of stx1 and stx2 by use of the TaqMan E. coli STX1 and STX2 PCR assays. Enrichments of samples positive for stx1 and/or stx2 were plated, and colony hybridization was performed using digoxigenin-labeled probes complementary to the stx1 and stx2 genes. Positive colonies were picked and confirmed by PCR for the presence of the stx1, stx2, or stx2e genes, and the isolates were serotyped. Out of 687 fecal samples tested using the TaqMan assays, 70% (484 of 687) were positive for Shiga toxin genes, and 54% (370 of 687), 64% (436 of 687), and 38% (261 of 687) were positive for stx1, stx2, and both toxin genes, respectively. Out of 219 isolates that were characterized, 29 (13%) produced stx1, 14 (6%) produced stx2, and 176 (80%) produced stx2e. Twenty-three fecal samples contained at least two STEC strains that had different serotypes but that had the same toxin genes or included a strain that possessed stx1 in addition to a strain that possessed stx2 or stx2e. The STEC isolates belonged to various serogroups, including O2, O5, O7, O8, O9, OX10, O11, O15, OX18, O20, O57, O65, O68, O69, O78, O91, O96, O100, O101, O120, O121, O152, O159, O160, O163, and O untypeable. It is noteworthy that no isolates of serogroup O157 were recovered. Results of this study indicate that swine in the United States harbor STEC that can potentially cause human illness. PMID:15574914

  3. MicroRNA Expression Profiling of Human Respiratory Epithelium Affected by Invasive Candida Infection.

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    Syed Aun Muhammad

    Full Text Available Invasive candidiasis is potentially life-threatening systemic fungal infection caused by Candida albicans (C. albicans. Candida enters the blood stream and disseminate throughout the body and it is often observed in hospitalized patients, immunocompromised individuals or those with chronic diseases. This infection is opportunistic and risk starts with the colonization of C. albicans on mucocutaneous surfaces and respiratory epithelium. MicroRNAs (miRNAs are small non-coding RNAs which are involved in the regulation of virtually every cellular process. They regulate and control the levels of mRNA stability and post-transcriptional gene expression. Aberrant expression of miRNAs has been associated in many disease states, and miRNA-based therapies are in progress. In this study, we investigated possible variations of miRNA expression profiles of respiratory epithelial cells infected by invasive Candida species. For this purpose, respiratory epithelial tissues of infected individuals from hospital laboratory were accessed before their treatment. Invasive Candida infection was confirmed by isolation of Candia albicans from the blood cultures of the same infected individuals. The purity of epithelial tissues was assessed by flow cytometry (FACSCalibur cytometer; BD Biosciences, Heidelberg, Germany using statin antibody (S-44. TaqMan quantitative real-time PCR (in a TaqMan Low Density Array format was used for miRNA expression profiling. MiRNAs investigated, the levels of expression of 55 miRNA were significantly altered in infected tissues. Some miRNAs showed dramatic increase (miR-16-1 or decrease of expression (miR-17-3p as compared to control. Gene ontology enrichment analysis of these miRNA-targeted genes suggests that Candidal infection affect many important biological pathways. In summary, disturbance in miRNA expression levels indicated the change in cascade of pathological processes and the regulation of respiratory epithelial functions

  4. Associations of Genetic Polymorphisms Relevant to Metabolic Pathway of Vitamin D3 with Development and Prognosis of Childhood Bronchial Asthma.

    Science.gov (United States)

    Zhang, Ying; Wang, Zhansheng; Ma, Tongshuai

    2017-08-01

    This study was aimed at investigating the correlation between genetic polymorphisms relevant to metabolic pathway of vitamin D3 (VD 3 ) and susceptibility to childhood bronchial asthma. Altogether 143 childhood patients with bronchial asthma and 143 healthy children of Chinese Han ethnicity were enrolled in this study. The key single-nucleotide polymorphisms (SNPs) were identified by HaploView 4.2 software and selected from previous investigations. Genomic DNAs were isolated from peripheral blood samples by using TaqMan Blood DNA kits. The genotyping of SNPs was performed by TaqMan SNPs genotyping assay. Odds ratios and corresponding 95% confidence intervals were calculated to evaluate the association between SNPs and susceptibility to bronchial asthma. Statistical analyses were conducted by using SPSS 13.0 software. Rs10766197 of CYP2R1, rs7041 and rs4588 of CG, rs4646536 of CYP27B1, rs2228570, rs7975232, and rs1544410 of VDR, as well as rs1805192 and rs10865710 of PPAR were shown to be significantly associated with increased risk of bronchial asthma. Besides, prognosis of childhood bronchial asthma, which was represented as Saint George Respiratory Questionnaire (SGRQ) scoring, was closely linked with CYP2R1 rs10766197, CYP27B1 rs4646536, VDR rs7975232, VDR rs1544410, PPAR rs1805192, and PPAR rs10865710. The haplotype analysis suggested that TA and CG of CG rs7041/rs4588, CA and AG of VDR rs7975232/rs1544410, and CC of PPAR rs1805192/rs10865710 were, respectively, correlated with levels of VD, IL-4, and IL-5. And only haplotypes of VDR showed associations with risk of bronchial asthma during childhood, whereas hardly any significance could be observed between the haplotypes and behavior of quality-of-life (SGRQ) scoring. Significant associations were found between rs10766197 of CYP2R1, rs7041 and rs4588 of CG, rs4646536 of CYP27B1, rs2228570, rs7975232, and rs1544410 of VDR, as well as rs1805192 and rs10865710 of PPAR and susceptibility to and prognosis of

  5. A novel mean-centering method for normalizing microRNA expression from high-throughput RT-qPCR data

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    Wylie Dennis

    2011-12-01

    Full Text Available Abstract Background Normalization is critical for accurate gene expression analysis. A significant challenge in the quantitation of gene expression from biofluids samples is the inability to quantify RNA concentration prior to analysis, underscoring the need for robust normalization tools for this sample type. In this investigation, we evaluated various methods of normalization to determine the optimal approach for quantifying microRNA (miRNA expression from biofluids and tissue samples when using the TaqMan® Megaplex™ high-throughput RT-qPCR platform with low RNA inputs. Findings We compared seven normalization methods in the analysis of variation of miRNA expression from biofluid and tissue samples. We developed a novel variant of the common mean-centering normalization strategy, herein referred to as mean-centering restricted (MCR normalization, which is adapted to the TaqMan Megaplex RT-qPCR platform, but is likely applicable to other high-throughput RT-qPCR-based platforms. Our results indicate that MCR normalization performs comparable to or better than both standard mean-centering and other normalization methods. We also propose an extension of this method to be used when migrating biomarker signatures from Megaplex to singleplex RT-qPCR platforms, based on the identification of a small number of normalizer miRNAs that closely track the mean of expressed miRNAs. Conclusions We developed the MCR method for normalizing miRNA expression from biofluids samples when using the TaqMan Megaplex RT-qPCR platform. Our results suggest that normalization based on the mean of all fully observed (fully detected miRNAs minimizes technical variance in normalized expression values, and that a small number of normalizer miRNAs can be selected when migrating from Megaplex to singleplex assays. In our study, we find that normalization methods that focus on a restricted set of miRNAs tend to perform better than methods that focus on all miRNAs, including

  6. Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas.

    Science.gov (United States)

    Butz, Henriett; Németh, Kinga; Czenke, Dóra; Likó, István; Czirják, Sándor; Zivkovic, Vladimir; Baghy, Kornélia; Korbonits, Márta; Kovalszky, Ilona; Igaz, Péter; Rácz, Károly; Patócs, Attila

    2017-07-01

    Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially

  7. Abundance of questing ticks and molecular evidence for pathogens in ticks in three parks of Emilia-Romagna region of Northern Italy

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    Sara Aureli

    2015-09-01

    Full Text Available Introduction and objective. Infectious and parasitic diseases transmitted by ticks, such as Lyme diseases, granulocytic anaplasmosis and piroplasmosis, have been frequently reported in Europe, with increasing attention to them as an emerging zoonotic problem. The presented study was performed to assess the distribution and the density of questing ticks in three regional parks of Emilia-Romagna region of Northern Italy, and to seek molecular evidence of potential human pathogens in tick populations. Materials and Methods. In the period April-October 2010, 8,139 questing ticks were collected: 6,734 larvae, 1,344 nymphs and only a few adults – 28 females and 33 males. The abundance of[i] Ixodes ricinus[/i] questing ticks was compared among different sampling sites and related to microclimate parameters. 1,544 out of 8,139 ticks were examined for the presence of pathogens: PCR was used to detect piroplasms DNA and Real time Taqman PCR for [i]Anaplasma phagocytophilum[/i] and [i]Borrelia burgdorferi[/i] s.l. Results. The predominant species was [i]I. ricinus[/i] (overall abundance 1,075.9/100 m[sup]2[/sup] ; more rarely, [i]Dermacentor marginatus[/i] (n = 37 – 0.45%, [i]Scaphixodes frontalis[/i] (n = 13 – 0.16%, [i]Hyalomma[/i] spp. (n = 6 – 0.07% and [i]Ixodes acuminatus[/i] (n = 3 – 0.04% were also found. 28 out of 324 (8.6% samples of ticks were PCR-positive for piroplasm DNA. 11 amplicons of 18S rRNA gene were identical to each other and had 100% identity with[i] Babesia[/i] EU1 ([i]Babesia venatorum[/i] using BLAST analysis. Real time Taqman PCR gave positive results for [i]A. phagocytophilum[/i] in 23 out of 292 samples (7.9%, and for [i]B. burgdorferi[/i] s.l. in 78 out of 292 samples (26.7%. [i]I. ricinu[/i]s was the only species found positive for pathogens by molecular analysis; 16 tick samples were co-infected with at least 2 pathogens. Discussion. The peak of nymph presence was in May, and the higher prevalence of pathogens

  8. Contrasting Plasmodium infection rates and insecticide susceptibility profiles between the sympatric sibling species Anopheles parensis and Anopheles funestus s.s: a potential challenge for malaria vector control in Uganda

    Science.gov (United States)

    2014-01-01

    Background Although the An. funestus group conceals one of the major malaria vectors in Africa, little is known about the dynamics of members of this group across the continent. Here, we investigated the species composition, infection rate and susceptibility to insecticides of this species group in Uganda. Methods Indoor resting blood-fed Anopheles adult female mosquitoes were collected from 3 districts in Uganda. Mosquitoes morphologically belonging to the An. funestus group were identified to species by PCR. The sporozoite infection rates were determined by TaqMan and a nested PCR. Susceptibility to major insecticides was assessed using WHO bioassays. The potential role of four candidate resistance genes was assessed using qRT-PCR. Results An. funestus s.s. and An. parensis, were the only members of the An. funestus group identified. Both species were sympatric in Masindi (North-West), whereas only An. parensis was present in Mityana (Central) and Ntungamo (South-West). The Plasmodium falciparum infection detected in An. parensis (4.2%) by TaqMan could not be confirmed by nested PCR, whereas the 5.3% infection in An. funestus s.s. was confirmed. An. parensis was susceptible to most insecticides, however, a moderate resistance was observed against deltamethrin and DDT. In the sympatric population of Masindi, resistance was observed to pyrethroids (permethrin and deltamethrin) and DDT, but all the resistant mosquitoes belonged to An. funestus s.s. No significant over-expression was observed for the four P450 candidate genes CYP6M7, CYP9K1, CYP6P9 and CYP6AA4 between deltamethrin resistant and control An. parensis. However, when compared with the susceptible FANG An. funestus s.s strain, the CYP9K1 is significantly over-expressed in An. parensis (15-fold change; P resistance. Conclusion The contrasting infection rates and insecticide susceptibility profiles of both species highlights the importance of accurate species identification for successful vector control

  9. Expression of microRNA related to bone remodeling regulation in plasma in patients with acromegaly

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    Tatiana A. Grebennikova

    2017-11-01

    Full Text Available Backgraund. MiсroRNA are small regulatory factors that regulate gene expression by post-transcriptional regulation of mRNA, playing an important role in numerous cellular processes including organogenesis, apoptosis, cell proliferation and differentiation. Acromegaly causes bone fragility, but the pathogenetic mechanism is generally unknown. Aim. To evaluate levels of microRNA related to bone remodeling regulation in plasma samples from patients with acromegaly Materials and methods. Fasting plasma samples were taken and stored in aliquot at ≤ -80°C from consecutive subjects with clinically evident and biochemically confirmed active acromegaly and healthy volunteers matched by age, sex and body mass index (BMI. miRNeasy Serum/Plasma Kit, TaqMan Advanced miRNA cDNA Synthesis Kit, TaqMan Advanced miRNA Assays were used to assay plasma miRNA expression. Insulin-like growth factor 1 (IGF1 was measured by immunochemiluminescence assay (Liaison. Results. We enrolled 40 subjects 22 patients suffered from acromegaly and 18 matched healthy controls matched by sex, age and BMI. The median age of patients with acromegaly was 42 years (Q25;Q75 – 37;43 with no difference among the groups, p=0.205; BMI – 28 (24;32 kg/m2, p=0.253. The median IGF1 in subjects with acromegaly – 622 (514;1000 ng/ml was significantly higher as compared to the control group (p<0.001. Patients with acromegaly had significantly higher expression of microRNA-100-5р (p=0.051, microRNA-550а-5р (p=0.048, microRNA-7b-5р (p=0.005 and microRNA-96-5р (p=0.042 among 27 bone-specific microRNA tested in plasma Conclusions. This study reveals that several microRNAs, known to regulate bone remodeling can be detected in plasma samples of patients with acromegaly and may be suggested as biomarkers for skeletal involvement in patients with acromegaly.

  10. Absolute estimation of initial concentrations of amplicon in a real-time RT-PCR process

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    Kohn Michael

    2007-10-01

    Full Text Available Abstract Background Since real time PCR was first developed, several approaches to estimating the initial quantity of template in an RT-PCR reaction have been tried. While initially only the early thermal cycles corresponding to exponential duplication were used, lately there has been an effort to use all of the cycles in a PCR. The efforts have included both fitting empirical sigmoid curves and more elaborate mechanistic models that explore the chemical reactions taking place during each cycle. The more elaborate mechanistic models require many more parameters than can be fit from a single amplification, while the empirical models provide little insight and are difficult to tailor to specific reactants. Results We directly estimate the initial amount of amplicon using a simplified mechanistic model based on chemical reactions in the annealing step of the PCR. The basic model includes the duplication of DNA with the digestion of Taqman probe and the re-annealing between previously synthesized DNA strands of opposite orientation. By modelling the amount of Taqman probe digested and matching that with the observed fluorescence, the conversion factor between the number of fluorescing dye molecules and observed fluorescent emission can be estimated, along with the absolute initial amount of amplicon and the rate parameter for re-annealing. The model is applied to several PCR reactions with known amounts of amplicon and is shown to work reasonably well. An expanded version of the model allows duplication of amplicon without release of fluorescent dye, by adding 1 more parameter to the model. The additional process is helpful in most cases where the initial primer concentration exceeds the initial probe concentration. Software for applying the algorithm to data may be downloaded at http://www.niehs.nih.gov/research/resources/software/pcranalyzer/ Conclusion We present proof of the principle that a mechanistically based model can be fit to observations

  11. Dicer and miRNA in relation to clinicopathological variables in colorectal cancer patients

    International Nuclear Information System (INIS)

    Stratmann, Johannes; Wang, Chao-Jie; Gnosa, Sebastian; Wallin, Åsa; Hinselwood, David; Sun, Xiao-Feng; Zhang, Hong

    2011-01-01

    Dicer is aberrantly expressed in several types of cancers. Applying real-time PCR, we detected the expression of Dicer mRNA in normal mucosa (n = 162), primary colorectal cancer (CRC) (n = 162) and liver metastasis (n = 37), and analysed the relationship between Dicer expression and clinicopathological features. We also correlated the expression of Dicer mRNA to the miRNA expression of miR-141, miR-200a, miR-200b, mir-200c and miR-429 in liver metastases. RT-PCR and qPCR were used to analyse the Dicer expression in normal mucosa, primary tumour and liver metastasis by using the High Capacity cDNA Reverse Transcription Kit and TaqMan™ ® Gene Expression assays for Dicer and GAPDH. RT-PCR and qPCR were used to detect miRNA expression in liver metastases by utilizing TaqMan ® MicroRNA Reverse Transcription Kit and TaqMan ® miRNA Assays. Statistical analyses were performed with STATISTICA. Dicer expression in rectal cancer (3.146 ± 0.953) was higher than in colon cancer (2.703 ± 1.204, P = 0.018). Furthermore the Dicer expression was increased in primary tumours (3.146 ± 0.952) in comparison to that in normal mucosa from rectal cancer patients (2.816 ± 1.009, P = 0.034) but this is not evident in colon cancer patients. Dicer expression in liver metastases was decreased in comparison to that of either normal mucosa or primary tumour in both colon and rectal cancers (P < 0.05). Patients with a high Dicer expression in normal mucosa had a worse prognosis compared to those with a low Dicer expression, independently of gender, age, tumour site, stage and differentiation (P < 0.001, RR 3.682, 95% CI 1.749 - 7.750). In liver metastases, Dicer was positively related to miR-141 (R = 0.419, P = 0.015). Dicer is up-regulated in the early development of rectal cancers. An increased expression of Dicer mRNA in normal mucosa from CRC patients is significantly related to poor survival independently of gender, age, tumour site, stage and differentiation

  12. Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

    Science.gov (United States)

    Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

    2014-07-05

    The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the

  13. Association of hypoxia inducible factor-1 alpha gene polymorphism with both type 1 and type 2 diabetes in a Caucasian (Hungarian sample

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    Panczel Pal

    2009-08-01

    Full Text Available Abstract Background Hypoxia inducible factor-1 alpha (HIF-1α is a transcription factor that plays an important role in neo-vascularisation, embryonic pancreas beta-cell mass development, and beta cell protection. Recently a non synonymous single nucleotide polymorphism (g.C45035T SNP, rs11549465 of HIF-1α gene, resulting in the p.P582S amino acid change has been shown to be associated with type 2 diabetes (T2DM in a Japanese population. Our aim was to replicate these findings on a Caucasian (Hungarian population, as well as to study whether this genetic effect is restricted to T2DM or can be expanded to diabetes in general. Methods A large Caucasian sample (N = 890 was recruited including 370 T2DM, 166 T1DM and 354 healthy subjects. Genotyping was validated by two independent methods: a restriction fragment analysis (RFLP and a real time PCR using TaqMan probes. An overestimation of heterozygotes by RFLP was observed as a consequence of a nearby SNP (rs34005929. Therefore genotyping results of the justified TaqMan system were accepted. The measured genotype distribution corresponded to Hardy-Weinberg equilibrium (P = 0.740 Results As the TT genotype was extremely rare in the population (0.6% in clinical sample and 2.5% in controls, the genotypes were grouped as T absent (CC and T present (CT and TT. Genotype-wise analysis showed a significant increase of T present group in controls (24.0% as compared to patients (16.8%, P = 0.008. This genetic effect was demonstrated in the separated samples of type 1 (15.1%, P = 0.020, and also in type 2 (17.6%, P = 0.032 diabetes. Allele-wise analysis gave identical results showing a higher frequency of the T allele in the control sample (13.3% than in the clinical sample (8.7%, P = 0.002 with similar results in type 1 (7.8%, P = 0.010 and type 2 (9.1%, P = 0.011 diabetes. The odds ratio for diabetes (either type 1 or 2 was 1.56 in the presence of the C allele. Conclusion We confirmed the protective effect

  14. Percutaneous bone cement refixation of aseptically loose hip prostheses: the effect of interface tissue removal on injected cement volumes

    Energy Technology Data Exchange (ETDEWEB)

    Malan, Daniel F. [Leiden University Medical Center, Department of Orthopaedics, Leiden (Netherlands); Delft University of Technology, Department of Intelligent Systems, Delft (Netherlands); Valstar, Edward R. [Leiden University Medical Center, Department of Orthopaedics, Leiden (Netherlands); Delft University of Technology, Department of Biomechanical Engineering, Delft (Netherlands); Nelissen, Rob G.H.H. [Leiden University Medical Center, Department of Orthopaedics, Leiden (Netherlands)

    2014-11-15

    To quantify whether injected cement volumes differed between two groups of patients who underwent experimental minimally invasive percutaneous cement injection procedures to stabilize aseptically loose hip prostheses. One patient group was preoperatively treated using gene-directed enzyme prodrug therapy to remove fibrous interface tissue, while the other group received no preoperative treatment. It was hypothesized that cement penetration may have been inhibited by the presence of fibrous interface tissue in periprosthetic lesions. We analyzed 17 patients (14 female, 3 male, ages 72-91, ASA categories 2-4) who were treated at our institution. Osteolytic lesions and injected cement were manually delineated using 3D CT image segmentation, and the deposition of injected cement was quantified. Patients who underwent preoperative gene-directed enzyme therapy to remove fibrous tissue exhibited larger injected cement volumes than those who did not. The observed median increase in injected cement volume was 6.8 ml. Higher cement leakage volumes were also observed for this group. We conclude that prior removal of periprosthetic fibrous interface tissue may enable better cement flow and penetration. This might lead to better refixation of aseptically loosened prostheses. (orig.)

  15. Comparison of Gene Expression in Human Embryonic Stem Cells, hESC-Derived Mesenchymal Stem Cells and Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Barbet, Romain; Peiffer, Isabelle; Hatzfeld, Antoinette; Charbord, Pierre; Hatzfeld, Jacques A

    2011-01-01

    We present a strategy to identify developmental/differentiation and plasma membrane marker genes of the most primitive human Mesenchymal Stem Cells (hMSCs). Using sensitive and quantitative TaqMan Low Density Arrays (TLDA) methodology, we compared the expression of 381 genes in human Embryonic Stem Cells (hESCs), hESC-derived MSCs (hES-MSCs), and hMSCs. Analysis of differentiation genes indicated that hES-MSCs express the sarcomeric muscle lineage in addition to the classical mesenchymal lineages, suggesting they are more primitive than hMSCs. Transcript analysis of membrane antigens suggests that IL1R1(low), BMPR1B(low), FLT4(low), LRRC32(low), and CD34 may be good candidates for the detection and isolation of the most primitive hMSCs. The expression in hMSCs of cytokine genes, such as IL6, IL8, or FLT3LG, without expression of the corresponding receptor, suggests a role for these cytokines in the paracrine control of stem cell niches. Our database may be shared with other laboratories in order to explore the considerable clinical potential of hES-MSCs, which appear to represent an intermediate developmental stage between hESCs and hMSCs.

  16. Genetic variations in the vitamin-D receptor (VDR) gene in preeclampsia patients in the Chinese Han population.

    Science.gov (United States)

    Zhan, Ying; Liu, Mengchun; You, Yuelan; Zhang, Yan; Wang, Jingli; Wang, Xunfeng; Liu, Shiguo; Liu, Xuemei

    2015-07-01

    Previous studies have indicated that vitamin D deficiency is linked to a risk of preeclampsia (PE). The aim of our study was to investigate the association between genetic variations in the vitamin-D receptor (VDR) gene and the susceptibility to PE in the Chinese Han population. We examined the genotypes VDR rs2228570, rs11568820 and rs1544410 in 402 PE patients and 554 normal pregnant women in the third trimester by TaqMan allelic discrimination real-time polymerase chain reaction. The clinical data of the individuals were collected to enable genotype-phenotype analysis. A significant statistical difference in the genotypic frequencies of rs2228570 between cases and controls was found (χ(2)=13.750, P=0.001). The G allele was the risk factor for the risk of PE (χ(2)=9.456, P=0.002, OR=1.137, 95% CI 1.111-1.610). There was no difference in the genotypic and allelic distributions of rs11568820 and rs1544410 between the two groups (P> 0.05). Our results provide evidence for a possible link between VDR and the development of PE in the Chinese Han population.

  17. Genetic variation in a microRNA-502 minding site in SET8 gene confers clinical outcome of non-small cell lung cancer in a Chinese population.

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    Jiali Xu

    Full Text Available BACKGROUND: Genetic variants may influence microRNA-target interaction through modulate their binding affinity, creating or destroying miRNA-binding sites. SET8, a member of the SET domain-containing methyltransferase, has been implicated in a variety array of biological processes. METHODS: Using Taqman assay, we genotyped a polymorphism rs16917496 T>C within the miR-502 binding site in the 3'-untranslated region of the SET8 gene in 576 non-small cell lung cancer (NSCLC patients. Functions of rs16917496 were investigated using luciferase activity assay and validated by immunostaining. RESULTS: Log-rank test and cox regression indicated that the CC genotype was associated with a longer survival and a reduced risk of death for NSCLC [58.0 vs. 41.0 months, P = 0.031; hazard ratio = 0.44, 95% confidential interval: 0.26-0.74]. Further stepwise regression analysis suggested rs16917496 was an independently favorable factor for prognosis and the protective effect more prominent in never smokers, patients without diabetes and patients who received chemotherapy. A significant interaction was observed between rs16917496 and smoking status in relation to NSCLC survival (PC located at miR-502 binding site contributes to NSCLC survival by altering SET8 expression through modulating miRNA-target interaction.

  18. FY 2000 Project of developing international standards for supporting new industries. Standardization of systems for detecting gene recombination products; 2000 nendo shinki sangyo shiengata kokusai hyojunka kaihatsu jigyo seika hokokusho. Idenshi kumikae seihin kenshutsu system no hyojunka

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-03-01

    Described herein are the FY 2000 results of the studies on the basic conditions for gene recombination products, and methods of determining content of the purchased gene recombination products in agricultural products, conducted to promote the standardization of the gene recombination product detection systems. Soybean is taken up as the gene recombination product to be studied, in which the samples containing the gene recombination product at 5, 0.5, 0.1 and 0% are prepared, and qualitatively analyzed by the DNA extraction and PCR method. No band is detected. For the quantitative examination by the real-time PCR method, the SYBRGeen, Taqman and hybridization probe methods are used to study the PCR conditions. The sample DNA extracted by the DNA Extraction kit for GMO Detection Ver.2 from the sample containing the RoundupReady{sup TM} soybean is quantitatively analyzed by the real-time PCR method for GMO content, which has produced the results for promotion of the standardization. (NEDO)

  19. Difference between age-related macular degeneration and polypoidal choroidal vasculopathy in the hereditary contribution of the A69S variant of the age-related maculopathy susceptibility 2 gene (ARMS2).

    Science.gov (United States)

    Yanagisawa, Suiho; Kondo, Naoshi; Miki, Akiko; Matsumiya, Wataru; Kusuhara, Sentaro; Tsukahara, Yasutomo; Honda, Shigeru; Negi, Akira

    2011-01-01

    To investigate whether the A69S variant of the age-related maculopathy susceptibility 2 gene (ARMS2) has a different hereditary contribution in neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). We initially conducted a comparative genetic analysis of neovascular AMD and PCV, genotyping the ARMS2 A69S variant in 181 subjects with neovascular AMD, 198 subjects with PCV, and 203 controls in a Japanese population. Genotyping was conducted using TaqMan technology. Results were then integrated into a meta-analysis of previous studies representing an assessment of the association between the ARMS2 A69S variant and neovascular AMD and/or PCV, comprising a total of 3,828 subjects of Asian descent. The Q-statistic test was used to assess between-study heterogeneity. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using a fixed effects model. The genetic effect of the A69S variant was stronger in neovascular AMD (allelic summary OR=3.09 [95% CI, 2.71-3.51], fixed effects parchitecture of this phenotypically heterogeneous disorder.

  20. Clinical evaluation of β-tubulin real-time PCR for rapid diagnosis of dermatophytosis, a comparison with mycological methods.

    Science.gov (United States)

    Motamedi, Marjan; Mirhendi, Hossein; Zomorodian, Kamiar; Khodadadi, Hossein; Kharazi, Mahboobeh; Ghasemi, Zeinab; Shidfar, Mohammad Reza; Makimura, Koichi

    2017-10-01

    Following our previous report on evaluation of the beta tubulin real-time PCR for detection of dermatophytosis, this study aimed to compare the real-time PCR assay with conventional methods for the clinical assessment of its diagnostic performance. Samples from a total of 853 patients with suspected dermatophyte lesions were subjected to direct examination (all samples), culture (499 samples) and real-time PCR (all samples). Fungal DNA was extracted directly from clinical samples using a conical steel bullet, followed by purification with a commercial kit and subjected to the Taq-Man probe-based real-time PCR. The study showed that among the 499 specimens for which all three methods were used, 156 (31.2%), 128 (25.6%) and 205 (41.0%) were found to be positive by direct microscopy, culture and real-time PCR respectively. Real-time PCR significantly increased the detection rate of dermatophytes compared with microscopy (288 vs 229) with 87% concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of the real-time PCR was 87.5%, 85%, 66.5% and 95.2% respectively. Although real-time PCR performed better on skin than on nail samples, it should not yet fully replace conventional diagnosis. © 2017 Blackwell Verlag GmbH.

  1. A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

    Science.gov (United States)

    Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

    2017-11-15

    A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. A real-time PCR approach to detect predation on anchovy and sardine early life stages

    Science.gov (United States)

    Cuende, Elsa; Mendibil, Iñaki; Bachiller, Eneko; Álvarez, Paula; Cotano, Unai; Rodriguez-Ezpeleta, Naiara

    2017-12-01

    Recruitment of sardine (Sardina pilchardus Walbaum, 1792) and anchovy (Engraulis encrasicolus Linnaeus, 1758) is thought to be regulated by predation of their eggs and larvae. Predators of sardine and anchovy can be identified by visual taxonomic identification of stomach contents, but this method is time consuming, tedious and may underestimate predation, especially in small predators such as fish larvae. Alternatively, genetic tools may offer a more cost-effective and accurate alternative. Here, we have developed a multiplex real-time polymerase chain reaction (RT-PCR) assay based on TaqMan probes to simultaneously detect sardine and anchovy remains in gut contents of potential predators. The assay combines previously described and newly generated species-specific primers and probes for anchovy and sardine detection respectively, and allows the detection of 0,001 ng of target DNA (which corresponds to about one hundredth of the total DNA present in a single egg). We applied the method to candidate anchovy and sardine egg predators in the Bay of Biscay, Atlantic Mackerel (Scomber scombrus) larvae. Egg predation observed was limited primarily to those stations where sardine and/or anchovy eggs were present. Our developed assay offers a suitable tool to understand the effects of predation on the survival of anchovy and sardine early life stages.

  3. Comparative analysis of minimal residual disease detection by multiparameter flow cytometry and enhanced ASO RQ-PCR in multiple myeloma

    International Nuclear Information System (INIS)

    Silvennoinen, R; Lundan, T; Kairisto, V; Pelliniemi, T-T; Putkonen, M; Anttila, P; Huotari, V; Mäntymaa, P; Siitonen, S; Uotila, L; Penttilä, T-L; Juvonen, V; Selander, T; Remes, K

    2014-01-01

    Multiparameter flow cytometry (MFC) and allele-specific oligonucleotide real-time quantitative PCR (ASO RQ-PCR) are the two most sensitive methods to detect minimal residual disease (MRD) in multiple myeloma (MM). We compared these methods in 129 paired post-therapy samples from 22 unselected, consecutive MM patients in complete/near complete remission. Appropriate immunophenotypic and ASO RQ-PCR-MRD targets could be detected and MRD analyses constructed for all patients. The high PCR coverage could be achieved by gradual widening of the primer sets used for clonality detection. In addition, for 13 (55%) of the patients, reverse orientation of the ASO primer and individual design of the TaqMan probe improved the sensitivity and specificity of ASO RQ-PCR analysis. A significant nonlinear correlation prevailed between MFC-MRD and PCR-MRD when both were positive. Discordance between the methods was found in 32 (35%) paired samples, which were negative by MFC-MRD, but positive by ASO RQ-PCR. The findings suggest that with the described technique, ASO RQ-PCR can be constructed for all patients with MM. ASO RQ-PCR is slightly more sensitive in MRD detection than 6−10-color flow cytometry. Owing to technical demands ASO RQ-PCR could be reserved for patients in immunophenotypic remission, especially in efficacy comparisons between different drugs and treatment modalities

  4. Reduction in PSA messenger-RNA expression and clinical recurrence in patients with prostatic cancer undergoing neoadjuvant therapy before radical prostatectomy

    Directory of Open Access Journals (Sweden)

    Baruffi Marco

    2004-04-01

    Full Text Available Abstract Background We assessed the incidence of micro-metastases at surgical margins (SM and pelvic lymph nodes (LN in patients submitted to radical retropubic prostatectomy (RP after neoadjuvant therapy (NT or to RP alone. We compared traditional staging to molecular detection of PSA using Taqman-based quantitative real-time PCR (qrt-PCR never used before for this purpose. Methods 29 patients were assigned to NT plus RP (arm A or RP alone (arm B. Pelvic LN were dissected for qrt-PCR analysis, together with right and left lateral SM. Results 64,3% patients of arm B and 26.6% of arm A had evidence of PSA mRNA expression in LN and/or SM. 17,2% patients, all of arm B, had biochemical recurrence. Conclusions Qrt-PCR may be more sensitive, compared to conventional histology, in identifying presence of viable prostate carcinoma cells in SM and LN. Gene expression of PSA in surgical periprostatic samples might be considered as a novel and reliable indicator of minimal residual disease after NT.

  5. Reduction in PSA messenger-RNA expression and clinical recurrence in patients with prostatic cancer undergoing neoadjuvant therapy before radical prostatectomy

    Science.gov (United States)

    Grasso, Marco; Lania, Caterina; Blanco, Salvatore; Baruffi, Marco; Mocellin, Simone

    2004-01-01

    Background We assessed the incidence of micro-metastases at surgical margins (SM) and pelvic lymph nodes (LN) in patients submitted to radical retropubic prostatectomy (RP) after neoadjuvant therapy (NT) or to RP alone. We compared traditional staging to molecular detection of PSA using Taqman-based quantitative real-time PCR (qrt-PCR) never used before for this purpose. Methods 29 patients were assigned to NT plus RP (arm A) or RP alone (arm B). Pelvic LN were dissected for qrt-PCR analysis, together with right and left lateral SM. Results 64,3% patients of arm B and 26.6% of arm A had evidence of PSA mRNA expression in LN and/or SM. 17,2% patients, all of arm B, had biochemical recurrence. Conclusions Qrt-PCR may be more sensitive, compared to conventional histology, in identifying presence of viable prostate carcinoma cells in SM and LN. Gene expression of PSA in surgical periprostatic samples might be considered as a novel and reliable indicator of minimal residual disease after NT. PMID:15104791

  6. Development of a qPCR Method for the Identification and Quantification of Two Closely Related Tuna Species, Bigeye Tuna (Thunnus obesus) and Yellowfin Tuna (Thunnus albacares), in Canned Tuna.

    Science.gov (United States)

    Bojolly, Daline; Doyen, Périne; Le Fur, Bruno; Christaki, Urania; Verrez-Bagnis, Véronique; Grard, Thierry

    2017-02-01

    Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, as juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional substitutions may occur during the canning process. In this study, two mitochondrial markers from NADH dehydrogenase subunit 2 and cytochrome c oxidase subunit II genes were used to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate these two closely related tuna species when used in a binary mix in tuna cans.

  7. Evaluation of candidate stromal epithelial cross-talk genes identifies association between risk of serous ovarian cancer and TERT, a cancer susceptibility "hot-spot".

    Directory of Open Access Journals (Sweden)

    Sharon E Johnatty

    2010-07-01

    Full Text Available We hypothesized that variants in genes expressed as a consequence of interactions between ovarian cancer cells and the host micro-environment could contribute to cancer susceptibility. We therefore used a two-stage approach to evaluate common single nucleotide polymorphisms (SNPs in 173 genes involved in stromal epithelial interactions in the Ovarian Cancer Association Consortium (OCAC. In the discovery stage, cases with epithelial ovarian cancer (n=675 and controls (n=1,162 were genotyped at 1,536 SNPs using an Illumina GoldenGate assay. Based on Positive Predictive Value estimates, three SNPs-PODXL rs1013368, ITGA6 rs13027811, and MMP3 rs522616-were selected for replication using TaqMan genotyping in up to 3,059 serous invasive cases and 8,905 controls from 16 OCAC case-control studies. An additional 18 SNPs with Pper-alleleor=0.5. However genotypes at TERT rs7726159 were associated with ovarian cancer risk in the smaller, five-study replication study (Pper-allele=0.03. Combined analysis of the discovery and replication sets for this TERT SNP showed an increased risk of serous ovarian cancer among non-Hispanic whites [adj. ORper-allele 1.14 (1.04-1.24 p=0.003]. Our study adds to the growing evidence that, like the 8q24 locus, the telomerase reverse transcriptase locus at 5p15.33, is a general cancer susceptibility locus.

  8. Comprehensive analysis of RET common and rare variants in a series of Spanish Hirschsprung patients confirms a synergistic effect of both kinds of events

    Directory of Open Access Journals (Sweden)

    Castaño Luis

    2011-10-01

    Full Text Available Abstract Background RET is the major gene associated to Hirschsprung disease (HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In the present study, we have performed a comprehensive study of our HSCR series evaluating the involvement of both RET rare variants (RVs and common variants (CVs in the context of the disease. Methods RET mutational screening was performed by dHPLC and direct sequencing for the identification of RVs. In addition Taqman technology was applied for the genotyping of 3 RET CVs previously associated to HSCR, including a variant lying in an enhancer domain within RET intron 1 (rs2435357. Statistical analyses were performed using the SPSS v.17.0 to analyze the distribution of the variants. Results Our results confirm the strongest association to HSCR for the "enhancer" variant, and demonstrate a significantly higher impact of it in male versus female patients. Integration of the RET RVs and CVs analysis showed that in 91.66% of cases with both kinds of mutational events, the enhancer allele is in trans with the allele bearing the RET RV. Conclusions A gender effect exists on both the transmission and distribution of rare coding and common HSCR causing mutations. In addition, these RET CVs and RVs seem to act in a synergistic way leading to HSCR phenotype.

  9. Prevalence of IFNL3 gene polymorphism among blood donors and its relation to genomic profile of ancestry in Brazil.

    Science.gov (United States)

    Rizzo, Silvia Renata Cornelio Parolin; Gazito, Diana; Pott-Junior, Henrique; Latini, Flavia Roche Moreira; Castelo, Adauto

    The recent development of interferon-free regimens based on direct-acting antivirals for the treatment of chronic hepatitis C virus infection has benefited many but not all patients. Some patients still experience treatment failure, possibly attributed to unknown host and viral factors, such as IFNL3 gene polymorphism. The present study assessed the prevalence of rs12979860-CC, rs12979860-CT, and rs12979860-TT genotypes of the IFNL3 gene, and its relationship with ancestry informative markers in 949 adult Brazilian healthy blood donors. Race was analyzed using ancestry informative markers as a surrogate for ancestry. IFNL3 gene was genotyped using the ABI TaqMan single nucleotide polymorphisms genotyping assays. The overall frequency of rs12979860-CC genotype was 36.9%. The contribution of African ancestry was significantly higher among donors from the northeast region in relation to southeast donors, whereas the influence of European ancestry was significantly higher in southeast donors. Donors with rs12979860-CC and rs12979860-CT genotypes had similar ancestry background. The contribution of African ancestry was higher among rs12979860-TT genotype donors in comparison to both rs12979860-CC and rs12979860-CT genotypes. The prevalence of rs12979860-CC genotype is similar to that found in the US, despite the Brazilian ancestry informative markers admixture. However, in terms of ancestry, rs12979860-CT genotype was much closer to rs12979860-CC individuals than to rs12979860-TT. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

  10. Altered Gene Expression by Low-Dose Arsenic Exposure in Humans and Cultured Cardiomyocytes: Assessment by Real-Time PCR Arrays

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    Judy Mumford

    2011-06-01

    Full Text Available Chronic arsenic exposure results in higher risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. The purpose of this study was to investigate the effects on expression of selected genes in the blood lymphocytes from 159 people exposed chronically to arsenic in their drinking water using a novel RT-PCR TaqMan low-density array (TLDA. We found that expression of tumor necrosis factor-α (TNF-α, which activates both inflammation and NF-κB-dependent survival pathways, was strongly associated with water and urinary arsenic levels. Expression of KCNA5, which encodes a potassium ion channel protein, was positively associated with water and toe nail arsenic levels. Expression of 2 and 11 genes were positively associated with nail and urinary arsenic, respectively. Because arsenic exposure has been reported to be associated with long QT intervals and vascular disease in humans, we also used this TLDA for analysis of gene expression in human cardiomyocytes exposed to arsenic in vitro. Expression of the ion-channel genes CACNA1, KCNH2, KCNQ1 and KCNE1 were down-regulated by 1-mM arsenic. Alteration of some common pathways, including those involved in oxidative stress, inflammatory signaling, and ion-channel function, may underlay the seemingly disparate array of arsenic-associated diseases, such as cancer, cardiovascular disease, and diabetes.

  11. Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

    Science.gov (United States)

    Malandraki, Ioanna; Beris, Despoina; Isaioglou, Ioannis; Olmos, Antonio; Varveri, Christina; Vassilakos, Nikon

    2017-01-01

    A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

  12. How conserved are the bacterial communities associated with aphids? A detailed assessment of the Brevicoryne brassicae (Hemiptera: Aphididae) using 16S rDNA.

    Science.gov (United States)

    Clark, E L; Daniell, T J; Wishart, J; Hubbard, S F; Karley, A J

    2012-12-01

    Aphids harbor a community of bacteria that include obligate and facultative endosymbionts belonging to the Enterobacteriaceae along with opportunistic, commensal, or pathogenic bacteria. This study represents the first detailed analysis of the identity and diversity of the bacterial community associated with the cabbage aphid, Brevicoryne brassicae (L.). 16S rDNA sequence analysis revealed that the community of bacteria associated with B. brassicae was diverse, with at least four different bacterial community types detected among aphid lines, collected from widely dispersed sites in Northern Britain. The bacterial sequence types isolated from B. brassicae showed little similarity to any bacterial endosymbionts characterized in insects; instead, they were closely related to free-living extracellular bacterial species that have been isolated from the aphid gut or that are known to be present in the environment, suggesting that they are opportunistic bacteria transmitted between the aphid gut and the environment. To quantify variation in bacterial community between aphid lines, which was driven largely by differences in the proportions of two dominant bacterial orders, the Pseudomonales and the Enterobacteriales, we developed a novel real-time (Taqman) qPCR assay. By improving our knowledge of aphid microbial ecology, and providing novel molecular tools to examine the presence and function of the microbial community, this study forms the basis of further research to explore the influence of the extracellular bacterial community on aphid fitness, pest status, and susceptibility to control by natural enemies.

  13. A small noncoding RNA signature found in exosomes of GBM patient serum as a diagnostic tool.

    Science.gov (United States)

    Manterola, Lorea; Guruceaga, Elizabeth; Gállego Pérez-Larraya, Jaime; González-Huarriz, Marisol; Jauregui, Patricia; Tejada, Sonia; Diez-Valle, Ricardo; Segura, Victor; Samprón, Nicolás; Barrena, Cristina; Ruiz, Irune; Agirre, Amaia; Ayuso, Angel; Rodríguez, Javier; González, Alvaro; Xipell, Enric; Matheu, Ander; López de Munain, Adolfo; Tuñón, Teresa; Zazpe, Idoya; García-Foncillas, Jesús; Paris, Sophie; Delattre, Jean Yves; Alonso, Marta M

    2014-04-01

    Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

  14. Survivin as a radioresistance factor in pancreatic cancer

    International Nuclear Information System (INIS)

    Asanuma, Koichi; Moriai, Ryosuke; Yajima, Tomomi; Yagihashi, Atsuhito; Yamada, Mikako; Kobayashi, Daisuke; Watanabe, Naoki

    2000-01-01

    We examined whether survivin acts as a constitutive and inducible radioresistance factor in pancreatic cancer cells. Using a quantitative TaqMan reverse transcription-polymerase chain reaction for survivin mRNA in five pancreatic cancer cell lines, we found an inverse relationship between survivin mRNA expression and radiosensitivity. PANC-1 cells, which had the highest survivin mRNA levels, were most resistant to X-irradiation; MIAPaCa-2 cells, which showed the least survivin mRNA expression, were the most sensitive to X-irradiation. Our results suggested that survivin could act as a constitutive radioresistance factor in pancreatic cancer cells. To determine whether radioresistance is enhanced by induction of survivin expression by irradiation, PANC-1 and MIAPaCa-2 cells were subjected to sublethal doses of X-irradiation followed by a lethal dose. Survivin mRNA expression was increased significantly in both PANC-1 and MIAPaCa-2 cell lines by pretreatment with a sublethal dose of X-irradiation, as was cell survival after exposure to the lethal dose. In this system, enzymatic caspase-3 activity was significantly suppressed in cells with acquired resistance. These results suggest that survivin also acts as an inducible radioresistance factor in pancreatic cancer cells. Survivin, then, appears to enhance radioresistance in pancreatic cancer cells; inhibition of survivin mRNA expression may improve the effectiveness of radiotherapy. (author)

  15. Real-time PCR method applied to seafood products for authentication of European sole (Solea solea) and differentiation of common substitute species.

    Science.gov (United States)

    Herrero, Beatriz; Lago, Fátima C; Vieites, Juan M; Espiñeira, Montserrat

    2012-01-01

    Judged by quality and taste, the European sole (Solea solea) is considered one of the finest flatfish and is, thus, of considerable commercial value. In the present work, a specific fast real-time PCR was developed for the authentication of S. solea, i.e. to distinguish it from other related species and avoid substitution of this species, either deliberately or unintentionally. The method is based on a species-specific set of primers and MGB Taqman probe which amplifies a 116-bp fragment of the internal transcribed spacer 1 (ITS 1) ribosomal DNA region. This assay combines the high specificity and sensitivity of real-time PCR with the rapidity of the fast mode, allowing the detection of S. solea in a short period of time. The present methodology was validated for application to all types of manufactured products for the presence of S. solea, with successful results. Subsequently, the method was applied to 40 commercial samples to determine whether correct labeling had been employed in the market. It was demonstrated that the assay is a useful tool in monitoring and verifying food labeling regulations.

  16. Epstein-Barr viral load before a liver transplant in children with chronic liver disease.

    Science.gov (United States)

    Shakibazad, Nader; Honar, Naser; Dehghani, Seyed Mohsen; Alborzi, Abdolvahab

    2014-12-01

    Many children with chronic liver disease require a liver transplant. These patients are prone to various infections, including Epstein-Barr virus infection. This study sought to measure the Epstein-Barr viral load by polymerase chain reaction before a liver transplant. This cross-sectional study was done at the Shiraz University of Medical Sciences, Shiraz, Iran, in 2011. All patients were aged younger than 18 years with chronic liver disease and were candidates for a liver transplant at the Shiraz Nemazee Hospital Organ Transplant Center. They had been investigated regarding their demographic characteristics, underlying disease, laboratory findings, and Epstein-Barr viral load by real-time TaqMan polymerase chain reaction. Ninety-eight patients were studied and the mean age was 6.5 ± 5.9 years. Cryptogenic cirrhosis was the most-prevalent reason for liver transplant, and the death rate before a transplant was 15%. Among the study subjects, 6 had measurable Epstein-Barr viral load by polymerase chain reaction before the transplant, and 4 of them had considerably higher Epstein-Barr viral loads (more than 1000 copies/mL). With respect to the close prevalence of posttransplant lymphoproliferative disease (6%) and the high Epstein-Barr viral load in the patients before a transplant (4%), high pretransplant Epstein-Barr viral load can be considered a risk factor for posttransplant lymphoproliferative disorder.

  17. Rapid Laboratory Identification of Neisseria meningitidis Serogroup C as the Cause of an Outbreak - Liberia, 2017.

    Science.gov (United States)

    Patel, Jaymin C; George, Josiah; Vuong, Jeni; Potts, Caelin C; Bozio, Catherine; Clark, Thomas A; Thomas, Jerry; Schier, Joshua; Chang, Arthur; Waller, Jessica L; Diaz, Maureen H; Whaley, Melissa; Jenkins, Laurel T; Fuller, Serena; Williams, Desmond E; Redd, John T; Arthur, Ray R; Taweh, Fahn; Vera Walker, Yatta; Hardy, Patrick; Freeman, Maxwell; Katawera, Victoria; Gwesa, Gulu; Gbanya, Miatta Z; Clement, Peter; Kohar, Henry; Stone, Mardia; Fallah, Mosoka; Nyenswah, Tolbert; Winchell, Jonas M; Wang, Xin; McNamara, Lucy A; Dokubo, E Kainne; Fox, LeAnne M

    2017-10-27

    On April 25, 2017, a cluster of unexplained illness and deaths among persons who had attended a funeral during April 21-22 was reported in Sinoe County, Liberia (1). Using a broad initial case definition, 31 cases were identified, including 13 (42%) deaths. Twenty-seven cases were from Sinoe County (1), and two cases each were from Grand Bassa and Monsterrado counties, respectively. On May 5, 2017, initial multipathogen testing of specimens from four fatal cases using the Taqman Array Card (TAC) assay identified Neisseria meningitidis in all specimens. Subsequent testing using direct real-time polymerase chain reaction (PCR) confirmed N. meningitidis in 14 (58%) of 24 patients with available specimens and identified N. meningitidis serogroup C (NmC) in 13 (54%) patients. N. meningitidis was detected in specimens from 11 of the 13 patients who died; no specimens were available from the other two fatal cases. On May 16, 2017, the National Public Health Institute of Liberia and the Ministry of Health of Liberia issued a press release confirming serogroup C meningococcal disease as the cause of this outbreak in Liberia.

  18. Lack of Association between ABO, PPAP2B, ADAMST7, PIK3CG, and EDNRA and Carotid Intima-Media Thickness, Carotid Plaques, and Cardiovascular Disease in Patients with Rheumatoid Arthritis

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    Raquel López-Mejías

    2014-01-01

    Full Text Available Introduction. Rheumatoid arthritis (RA is a polygenic disease associated with accelerated atherosclerosis and increased cardiovascular (CV mortality. Recent studies have identified the ABO rs579459, PPAP2B rs17114036, and ADAMTS7 rs3825807 polymorphisms as genetic variants associated with coronary artery disease and the PIK3CG rs17398575 and EDNRA rs1878406 polymorphisms as the most significant signals related to the presence of carotid plaque in nonrheumatic Caucasian individuals. Accordingly, we evaluated the potential relationship between these 5 polymorphisms and subclinical atherosclerosis (assessed by carotid intima-media thickness (cIMT and presence/absence of carotid plaques and CV disease in RA. Material and Methods. 2140 Spanish RA patients were genotyped for the 5 polymorphisms by TaqMan assays. Subclinical atherosclerosis was evaluated in 620 of these patients by carotid ultrasonography technology. Results. No statistically significant differences were found when each polymorphism was assessed according to cIMT values and presence/absence of carotid plaques in RA, after adjusting the results for potential confounders. Moreover, no significant differences were obtained when RA patients were stratified according to the presence/absence of CV disease after adjusting for potential confounders. Conclusion. Our results do not confirm association between ABO rs579459, PPAP2B rs17114036, ADAMTS7 rs3825807, PIK3CG rs17398575, and EDNRA rs1878406 and subclinical atherosclerosis and CV disease in RA.

  19. The impact of HLA-G, LILRB1 and LILRB2 gene polymorphisms on susceptibility to and severity of endometriosis.

    Science.gov (United States)

    Bylińska, Aleksandra; Wilczyńska, Karolina; Malejczyk, Jacek; Milewski, Łukasz; Wagner, Marta; Jasek, Monika; Niepiekło-Miniewska, Wanda; Wiśniewski, Andrzej; Płoski, Rafał; Barcz, Ewa; Roszkowski, Piotr; Kamiński, Paweł; Malinowski, Andrzej; Wilczyński, Jacek R; Radwan, Paweł; Radwan, Michał; Kuśnierczyk, Piotr; Nowak, Izabela

    2018-06-01

    Endometriosis is a disease in which endometriotic tissue occurs outside the uterus. Its pathogenesis is still unknown. The most widespread hypothesis claims that ectopic endometrium appears as a result of retrograde menstruation and its insufficient elimination by immunocytes. Some reports have shown expression of non-classical HLA-G molecules on ectopic endometrium. HLA-G is recognized by KIR2DL4, LILRB1 and LILRB2 receptors on natural killer (NK) and other cells. These receptors are polymorphic, which may affect their activity. In this study we investigated whether HLA-G, KIR2DL4, LILRB1 and LILRB2 polymorphisms may influence susceptibility to endometriosis and disease progression. We used polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and allelic discrimination methods with TaqMan SNP Genotyping Assays for typing of 276 patients with endometriosis and 314 healthy fertile women. The HLA-G rs1632947:GG genotype was associated with protection against the disease and its severe stages; HLA-G rs1233334:CT protected against progression; LILRB1 rs41308748:AA and LILRB2 rs383369:AG predisposed to the disease and its progression. No effect of KIR2DL4 polymorphism was observed. These results support the role of polymorphisms of HLA-G and its receptors LILRB1 and LILRB2 in susceptibility to endometriosis and its progression.

  20. Lack of association of variants previously associated with anti-TNF medication response in rheumatoid arthritis patients: results from a homogeneous Greek population.

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    Maria I Zervou

    Full Text Available Treatment strategies blocking tumor necrosis factor (anti-TNF have proven very successful in patients with rheumatoid arthritis (RA, showing beneficial effects in approximately 50-60% of the patients. However, a significant subset of patients does not respond to anti-TNF agents, for reasons that are still unknown. The aim of this study was to validate five single nucleotide polymorphisms (SNPs of PTPRC, CD226, AFF3, MyD88 and CHUK gene loci that have previously been reported to predict anti-TNF outcome. In addition, two markers of RA susceptibility, namely TRAF1/C5 and STAT4 were assessed, in a cohort of anti-TNF-treated RA patients, from the homogeneous Greek island of Crete, Greece. The RA patient cohort consisted of 183 patients treated with either of 3 anti-TNF biologic agents (infliximab, adalimumab and etanercept from the Clinic of Rheumatology of the University Hospital of Crete. The SNPs were genotyped by TaqMan assays or following the Restriction Fragments Length Polymorphisms (RFLPs approach. Disease activity score in 28 joints (DAS28 at baseline and after 6 months were available for all patients and analysis of good versus poor response at 6 months was performed for each SNP. None of the 7 genetic markers correlated with treatment response. We conclude that the gene polymorphisms under investigation are not strongly predictive of anti-TNF response in RA patients from Greece.

  1. Lack of association of variants previously associated with anti-TNF medication response in rheumatoid arthritis patients: results from a homogeneous Greek population.

    Science.gov (United States)

    Zervou, Maria I; Myrthianou, Efsevia; Flouri, Irene; Plant, Darren; Chlouverakis, Gregory; Castro-Giner, Francesc; Rapsomaniki, Panayiota; Barton, Anne; Boumpas, Dimitrios T; Sidiropoulos, Prodromos; Goulielmos, George N

    2013-01-01

    Treatment strategies blocking tumor necrosis factor (anti-TNF) have proven very successful in patients with rheumatoid arthritis (RA), showing beneficial effects in approximately 50-60% of the patients. However, a significant subset of patients does not respond to anti-TNF agents, for reasons that are still unknown. The aim of this study was to validate five single nucleotide polymorphisms (SNPs) of PTPRC, CD226, AFF3, MyD88 and CHUK gene loci that have previously been reported to predict anti-TNF outcome. In addition, two markers of RA susceptibility, namely TRAF1/C5 and STAT4 were assessed, in a cohort of anti-TNF-treated RA patients, from the homogeneous Greek island of Crete, Greece. The RA patient cohort consisted of 183 patients treated with either of 3 anti-TNF biologic agents (infliximab, adalimumab and etanercept) from the Clinic of Rheumatology of the University Hospital of Crete. The SNPs were genotyped by TaqMan assays or following the Restriction Fragments Length Polymorphisms (RFLPs) approach. Disease activity score in 28 joints (DAS28) at baseline and after 6 months were available for all patients and analysis of good versus poor response at 6 months was performed for each SNP. None of the 7 genetic markers correlated with treatment response. We conclude that the gene polymorphisms under investigation are not strongly predictive of anti-TNF response in RA patients from Greece.

  2. Characterization of polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 and relationship to the alcoholism in a Colombian population.

    Science.gov (United States)

    Méndez, Claudia; Rey, Mauricio

    2015-12-30

    Identify and characterize polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 in a Colombian population residing in the city of Bogotá and determine its possible relationship to the alcoholism. ADH2, ADH3, ALDH2, and CYP2E1 genotypes a population of 148 individuals with non-problematic alcohol and 65 individuals with alcoholism were determined with TaqMan probes and PCR-RFLP. DNA was obtained from peripheral blood white cells. Significant difference was found in family history of alcoholism and use of other psychoactive substances to compare alcoholics with controls. When allelic frequencies for each category (gender) were considered, frequency of A2 allele carriers in ADH2 was found higher in male patients than controls. In women, the relative frequency for c1 allele in CYP2E1 was lower in controls than alcoholics. The ALDH2 locus is monomorphic. No significant differences in allele distributions of the loci examined to compare two populations were observed, however when stratifying the same trend was found that these differences tended to be significant. This study allows us to conclude the positive association between family history of alcoholism and alcoholism suggesting that there is a favourable hereditary predisposition. Since substance dependence requires interaction of multiple genes, the combination of genotypes ADH2 * 2, CYP2E1 * 1 combined with genotype homozygous ALDH2 * 1 found in this study could be leading to the population to a potential risk to alcoholism.

  3. Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters.

    Science.gov (United States)

    Jenkins, M B; Endale, D M; Fisher, D S; Gay, P A

    2009-02-01

    To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method. The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0.1 MPN per litre, with a 95% confidence level of 0.01-0.7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0.9 MPN per litre for pond outflow and from not detectable to 8.3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from 10(4) MPN per hour. This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife. This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.

  4. Polymorphisms in FFAR4 (GPR120 Gene Modulate Insulin Levels and Sensitivity after Fish Oil Supplementation

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    Bastien Vallée Marcotte

    2017-11-01

    Full Text Available The objective was to test whether FFAR4 single nucleotide polymorphisms (SNPs are associated with glycemic control-related traits in humans following fish oil supplementation. A total of 210 participants were given 3 g/day of omega-3 (n-3 fatty acids (FA (1.9–2.2 g of eicosapentaenoic acid (EPA and 1.1 g of docosahexaenoic acid (DHA during six weeks. Biochemical parameters were taken before and after the supplementation. Using the HapMap database and the tagger procedure in Haploview, 12 tagging SNPs in FFAR4 were selected and then genotyped using TaqMan technology. Transcript expression levels were measured for 30 participants in peripheral mononuclear blood cells. DNA methylation levels were measured for 35 participants in leukocytes. In silico analyses were also performed. Four gene–diet interactions on fasting insulin levels and homeostatic model assessment of insulin resistance (HOMA-IR index values were found. rs17108973 explained a significant proportion of the variance of insulin levels (3.0% and HOMA-IR (2.03% index values. Splice site prediction was different depending on the allele for rs11187527. rs17108973 and rs17484310 had different affinity for transcription factors depending on the allele. n-3 FAs effectively improve insulin-related traits for major allele homozygotes of four FFAR4 SNPs as opposed to carriers of the minor alleles.

  5. Polymorphisms in Genes Involved in Fatty Acid β-Oxidation Interact with Dietary Fat Intakes to Modulate the Plasma TG Response to a Fish Oil Supplementation

    Science.gov (United States)

    Bouchard-Mercier, Annie; Rudkowska, Iwona; Lemieux, Simone; Couture, Patrick; Vohl, Marie-Claude

    2014-01-01

    A large inter-individual variability in the plasma triglyceride (TG) response to an omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs) within genes involved in fatty acid β-oxidation and dietary fat intakes. Two hundred and eight (208) participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA). Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187) and ACOX1 (rs17583163) genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation. PMID:24647074

  6. Polymorphisms in Genes Involved in Fatty Acid β-Oxidation Interact with Dietary Fat Intakes to Modulate the Plasma TG Response to a Fish Oil Supplementation

    Directory of Open Access Journals (Sweden)

    Annie Bouchard-Mercier

    2014-03-01

    Full Text Available A large inter-individual variability in the plasma triglyceride (TG response to an omega-3 polyunsaturated fatty acid (n-3 PUFA supplementation has been observed. The objective was to examine gene-diet interaction effects on the plasma TG response after a fish oil supplementation, between single-nucleotide polymorphisms (SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes. Two hundred and eight (208 participants were recruited in the greater Quebec City area. The participants completed a six-week fish oil supplementation (5 g fish oil/day: 1.9–2.2 g EPA and 1.1 g DHA. Dietary fat intakes were measured using three-day food records. SNPs within RXRA, CPT1A, ACADVL, ACAA2, ABCD2, ACOX1 and ACAA1 genes were genotyped using TAQMAN methodology. Gene-diet interaction effects on the plasma TG response were observed for SNPs within RXRA (rs11185660, rs10881576 and rs12339187 and ACOX1 (rs17583163 genes. For rs11185660, fold changes in RXRA gene expression levels were different depending on SFA intakes for homozygotes T/T. Gene-diet interaction effects of SNPs within genes involved in fatty acid β-oxidation and dietary fat intakes may be important in understanding the inter-individual variability in plasma TG levels and in the plasma TG response to a fish oil supplementation.

  7. Dobrava virus carried by the yellow-necked field mouse Apodemus flavicollis, causing hemorrhagic fever with renal syndrome in Romania.

    Science.gov (United States)

    Panculescu-Gatej, Raluca Ioana; Sirbu, Anca; Dinu, Sorin; Waldstrom, Maria; Heyman, Paul; Murariu, Dimitru; Petrescu, Angela; Szmal, Camelia; Oprisan, Gabriela; Lundkvist, Ake; Ceianu, Cornelia S

    2014-05-01

    Hemorrhagic fever with renal syndrome (HFRS) has been confirmed by serological methods during recent years in Romania. In the present study, focus-reduction neutralization tests (FRNT) confirmed Dobrava hantavirus (DOBV) as the causative agent in some HFRS cases, but could not distinguish between DOBV and Saaremaa virus (SAAV) infections in other cases. DOBV was detected by a DOBV-specific TaqMan assay in sera of nine patients out of 22 tested. Partial sequences of the M genomic segment of DOBV were obtained from sera of three patients and revealed the circulation of two DOBV lineages in Romania. Investigation of rodents trapped in Romania found three DOBV-positive Apodemus flavicollis out of 83 rodents tested. Two different DOBV lineages were also detected in A. flavicollis as determined from partial sequences of the M and S genomic segments. Sequences of DOBV in A. flavicollis were either identical or closely related to the sequences obtained from the HFRS patients. The DOBV strains circulating in Romania clustered in two monophyletic groups, together with strains from Slovenia and the north of Greece. This is the first evidence for the circulation of DOBV in wild rodents and for a DOBV etiology of HFRS in Romania.

  8. Improved Method for the Detection and Quantification of Naegleria fowleri in Water and Sediment Using Immunomagnetic Separation and Real-Time PCR

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    Bonnie J. Mull

    2013-01-01

    Full Text Available Primary amebic meningoencephalitis (PAM is a rare and typically fatal infection caused by the thermophilic free-living ameba, Naegleria fowleri. In 2010, the first confirmed case of PAM acquired in Minnesota highlighted the need for improved detection and quantification methods in order to study the changing ecology of N. fowleri and to evaluate potential risk factors for increased exposure. An immunomagnetic separation (IMS procedure and real-time PCR TaqMan assay were developed to recover and quantify N. fowleri in water and sediment samples. When one liter of lake water was seeded with N. fowleri strain CDC:V212, the method had an average recovery of 46% and detection limit of 14 amebas per liter of water. The method was then applied to sediment and water samples with unknown N. fowleri concentrations, resulting in positive direct detections by real-time PCR in 3 out of 16 samples and confirmation of N. fowleri culture in 6 of 16 samples. This study has resulted in a new method for detection and quantification of N. fowleri in water and sediment that should be a useful tool to facilitate studies of the physical, chemical, and biological factors associated with the presence and dynamics of N. fowleri in environmental systems.

  9. Association of gene polymorphism of the fat mass and obesity associated gene with metabolic syndrome: a retrospective cohort study in Japanese workers.

    Science.gov (United States)

    Kawajiri, Tomoka; Osaki, Yoneatsu; Kishimoto, Takuji

    2012-06-01

    To investigate whether gene polymorphism of the fat mass and obesity associated gene (FTO) is associated with metabolic syndrome (MS), we used two MS criteria, the National Cholesterol Education Program-Adult Treatment panel III (NCEP-ATPIII) definition in 2003 and the Japanese definition in 2005. Subjects were respectively 859 and 865 Japanese workers at a company in Shimane Prefecture, Japan. They were non-MS individuals in 1998 and had regular health checkups between 1998 and 2006. The Cox proportional hazard regression was used to predict MS. Three SNPs in the FTO, rs9939609, rs1121980 and rs1558902, were genotyped by the TaqMan PCR assay and a retrospective study was performed. The three SNPs in the FTO were significantly associated with body mass index, and rs1121980 and rs1558902 were associated with fasting plasma glucose. MS defined by the NCEP-ATPIII definition was significantly associated with additive and dominant models of rs9939609 and rs1121980, and the dominant model of rs1558902, even after adjusting for confounding factors such as age, sex and lifestyle. MS defined by the Japanese definition was significantly associated with the additive model of rs1121980 and additive and dominant models of rs1558902 in multivariate analysis. These results suggested that FTO gene polymorphisms, rs9939609, rs1121980 and rs1558902, were associated with an increased risk of MS among Japanese workers.

  10. Prevalence of Ambler class A β-lactamases and ampC expression in cephalosporin-resistant isolates of Acinetobacter baumannii.

    Science.gov (United States)

    Rezaee, Mohammad Ahangarzadeh; Pajand, Omid; Nahaei, Mohammad Reza; Mahdian, Reza; Aghazadeh, Mohammad; Ghojazadeh, Morteza; Hojabri, Zoya

    2013-07-01

    We examined the prevalence of various cephalosporins' resistance mechanisms in Acinetobacter baumannii clinical isolates. Phenotypic and molecular detection of Ambler classes A, B and D β-lactamases was performed on 75 isolates. Clonal relatedness was defined using Repetitive Extragenic Palindromic PCR. PCR mapping was used to examine the linkage of insertion sequences and the ampC gene, and ampC expression was analyzed by TaqMan reverse transcriptase-PCR. Twenty-six (37%) isolates carried at least one of the blaPER-1 or blaTEM-1. Sixty-nine (98.5%) out of 70 cephalosporin-resistant isolates had insertions upstream of the ampC gene, of which 48 (69%) and 6 (8%) were identified as ISAba1and ISAba125, respectively. Higher level of expression was obtained in resistant isolates lacking ISAba1/ampC combination in comparison with that in positive ones. The ability to up-regulate the expression of ampC gene in association with different insertion elements has become an important factor in A. baumannii resistance to cephalosporins. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Gender-dependent association of HSD11B1 single nucleotide polymorphisms with glucose and HDL-C levels

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    Luciane Viater Turek

    2014-09-01

    Full Text Available In this study, we investigated the influence of two SNPs (rs846910 and rs12086634 of the HSD11B1 gene that encodes 11β-hydroxysteroid dehydrogenase type 1(11β-HSD1, the enzyme that catalyzes the conversion of cortisol to cortisone, on variables associated with obesity and metabolic syndrome in 215 individuals of both sexes from southern Brazil. The HSD11B1 gene variants were genotyped using the TaqMan SNP genotyping assay. Glucose, triglycerides, total cholesterol, HDL-cholesterol and LDL-cholesterol were measured by standard automated methods. Significant results were found in women, with carriers of the G allele of SNP rs12086634 having higher glucose levels than non-carriers. Carriers of the A allele of SNP rs846910 had higher levels of HDL-cholesterol. The involvement of both polymorphisms as independent factors in determining the levels of glucose and HDL-cholesterol was confirmed by multiple regression analysis (β = 0.19 ± 0.09, p = 0.03 and β = 0.22 ± 0.10, p = 0.03, respectively. Our findings suggest that the HSD11B1SNPs studied may indirectly influence glucose and HDL-cholesterol metabolism in women, possibly through down-regulation of the HSD11B1 gene by estrogen.

  12. F4-related mutation and expression analysis of the aminopeptidase N gene in pigs.

    Science.gov (United States)

    Goetstouwers, T; Van Poucke, M; Nguyen, V U; Melkebeek, V; Coddens, A; Deforce, D; Cox, E; Peelman, L J

    2014-05-01

    Intestinal infections with F4 enterotoxigenic Escherichia coli (ETEC) are worldwide an important cause of diarrhea in neonatal and recently weaned pigs. Adherence of F4 ETEC to the small intestine by binding to specific receptors is mediated by F4 fimbriae. Porcine aminopeptidase N (ANPEP) was recently identified as a new F4 receptor. In this study, 7 coding mutations and 1 mutation in the 3' untranslated region (3' UTR)were identified in ANPEP by reverse transcriptase (RT-) PCR and sequencing using 3 F4 receptor-positive (F4R+) and 2 F4 receptor-negative (F4R-) pigs, which were F4 phenotyped based on the MUC4 TaqMan, oral immunization, and the in vitro villous adhesion assay. Three potential differential mutations (g.2615C > T, g.8214A > G, and g.16875C > G) identified by comparative analysis between the 3 F4R+ and 2 F4R- pigs were genotyped in 41 additional F4 phenotyped pigs. However, none of these 3 mutations could be associated with F4 ETEC susceptibility. In addition, the RT-PCR experiments did not reveal any differential expression or alternative splicing in the small intestine of F4R+ and F4R- pigs. In conclusion, we hypothesize that the difference in F4 binding to ANPEP is due to modifications in its carbohydrate moieties.

  13. Geographic and habitat partitioning of genetically distinct zooxanthellae (Symbiodinium) in Acropora corals on the Great Barrier Reef.

    Science.gov (United States)

    Ulstrup, K E; Van Oppen, M J H

    2003-12-01

    Intra- and intercolony diversity and distribution of zooxanthellae in acroporid corals is largely uncharted. In this study, two molecular methods were applied to determine the distribution of zooxanthellae in the branching corals Acropora tenuis and A. valida at several reef locations in the central section of the Great Barrier Reef. Sun-exposed and shaded parts of all colonies were examined. Single-stranded conformational polymorphism analysis showed that individual colonies of A. tenuis at two locations harbour two strains of Symbiodinium belonging to clade C (C1 and C2), whereas conspecific colonies at two other reefs harboured a single zooxanthella strain. A. valida was found to simultaneously harbour strains belonging to two distinct phylogenetic clades (C and D) at all locations sampled. A novel method with improved sensitivity (quantitative polymerase chain reaction using Taqman fluorogenic probes) was used to map the relative abundance distribution of the two zooxanthella clades. At two of the five sampling locations both coral species were collected. At these two locations, composition of the zooxanthella communities showed the same pattern in both coral species, i.e. correlation with ambient light in Pioneer Bay and an absence thereof in Nelly Bay. The results show that the distribution of genetically distinct zooxanthellae is correlated with light regime and possibly temperature in some (but not all) colonies of A. tenuis and A. valida and at some reef locations, which we interpret as acclimation to local environmental conditions.

  14. Rare Mutations of Peroxisome Proliferator-Activated Receptor Gamma: Frequencies and Relationship with Insulin Resistance and Diabetes Risk in the Mixed Ancestry Population from South Africa

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    Z. Vergotine

    2014-01-01

    Full Text Available Background. Genetic variants in the nuclear transcription receptor, PPARG, are associated with cardiometabolic traits, but reports remain conflicting. We determined the frequency and the clinical relevance of PPARG SNPs in an African mixed ancestry population. Methods. In a cross-sectional study, 820 participants were genotyped for rs1800571, rs72551362, rs72551363, rs72551364, and rs3856806, using allele-specific TaqMan technology. The homeostatic model assessment of insulin (HOMA-IR, β-cells function (HOMA-B%, fasting insulin resistance index (FIRI, and the quantitative insulin-sensitivity check index (QUICKI were calculated. Results. No sequence variants were found except for the rs3856806. The frequency of the PPARG-His447His variant was 23.8% in the overall population group, with no difference by diabetes status (P=0.215. The His447His allele T was associated with none of the markers of insulin resistance overall and by diabetes status. In models adjusted for 2-hour insulin, the T allele was associated with lower prevalent diabetes risk (odds ratio 0.56 (95% CI 0.31–0.95. Conclusion. Our study confirms the almost zero occurrences of known rare PPARG SNPs and has shown for the first time in an African population that one of the common SNPs, His447His, may be protective against type 2 diabetes.

  15. Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies.

    Science.gov (United States)

    Passot, Christophe; Azzopardi, Nicolas; Renault, Sylvaine; Baroukh, Nadine; Arnoult, Christophe; Ohresser, Marc; Boisdron-Celle, Michèle; Gamelin, Erick; Watier, Hervé; Paintaud, Gilles; Gouilleux-Gruart, Valérie

    2013-01-01

    The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman (®) technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs' distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.

  16. [Association of T174M polymorphism of the angiotensinogen gene with the higher risk of cerebral stroke in women].

    Science.gov (United States)

    Stetskaia, T A; Bushueva, O Iu; Bulgakova, I V; Vialykh, E K; Shuteeva, T V; Biriukov, A E; Ivanov, V P; Polonikov, A V

    2014-01-01

    To study the association of M235T (rs699) and T174M (rs4762) polymorphisms of the angiotensinogen (AGT) gene with the risk of cerebral stroke (CS) in the Russians of the Central Chernozem Region. A total of 638 DNA samples obtained from 353 patients with CS and 285 sex- and age-matched healthy individuals were examined. The polymorphisms were genotyped by polymerase chain reaction (T174M) and TaqMan allelic discrimination (M235T) assays. Heterozygous AGT 174TM genotype carriers were found to be at a higher risk for CS (odd ratio (OR) = 1.52; 95% confidence interval (CI), 1.08-2.15; p = 0.02). A gender-stratified analysis showed that the mutant 174M allele (OR = 1.86; 95% CI, 1.14-3.03, p = 0.01) and variant 174TM and 174MM genotypes (OR = 1.86; 95% CI, 1.09-3.20; p = 0.02) were associated with the higher risk of cerebral stroke in women. The association of AGT T174M polymorphism with the risk of CS was first found; but the higher risk of the disease in the carriers of variant alleles and genotypes was observed in the women only.

  17. A Simple Phosphate-Buffered-Saline-Based Extraction Method Improves Specificity of HIV Viral Load Monitoring Using Dried Blood Spots.

    Science.gov (United States)

    Makadzange, A Tariro; Boyd, F Kathryn; Chimukangara, Benjamin; Masimirembwa, Collen; Katzenstein, David; Ndhlovu, Chiratidzo E

    2017-07-01

    Although Roche COBAS Ampliprep/COBAS TaqMan (CAP/CTM) systems are widely used in sub-Saharan Africa for early infant diagnosis of HIV from dried blood spots (DBS), viral load monitoring with this system is not practical due to nonspecific extraction of both cell-free and cell-associated viral nucleic acids. A simplified DBS extraction technique for cell-free virus elution using phosphate-buffered saline (PBS) may provide an alternative analyte for lower-cost quantitative HIV virus load (VL) testing to monitor antiretroviral therapy (ART). We evaluated the CAP/CTM v2.0 assay in 272 paired plasma and DBS specimens using the cell-free virus elution method and determined the level of agreement, sensitivity, and specificity at thresholds of target not detected (TND), target below the limit of quantification (BLQ) (1,000 copies/ml, the sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) were 92.7%, 100%, 100%, and 94.3%, respectively. PBS elution of DBS offers a sensitive and specific method for monitoring plasma viremia among adults and children on ART at the WHO-recommended threshold of >1,000 copies/ml on the Roche CAP/CTM system. Copyright © 2017 Makadzange et al.

  18. Novel Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Varroa destructor Populations from the Southeastern USA.

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    González-Cabrera, Joel; Rodríguez-Vargas, Sonia; Davies, T G Emyr; Field, Linda M; Schmehl, Daniel; Ellis, James D; Krieger, Klemens; Williamson, Martin S

    2016-01-01

    The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V) of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes.

  19. Vascular Endothelial Growth Factor Receptor-2 Polymorphisms Have Protective Effect against the Development of Tendinopathy in Volleyball Athletes.

    Science.gov (United States)

    Salles, José Inácio; Duarte, Maria Eugenia Leite; Guimarães, João Matheus; Lopes, Lucas Rafael; Vilarinho Cardoso, Jessica; Aguiar, Diego Pinheiro; Machado Neto, João Olyntho; Machado, Daniel Escorsim; Perini, Jamila Alessandra

    2016-01-01

    The aim of the study was to investigate whether genetic variants in VEGF and KDR genes can be correlated with susceptibility of tendinopathy in volleyball athletes. This study was conducted at the Brazilian Volleyball Federation, and comprised 179 volleyball athletes: 88 had a confirmed diagnosis of tendinopathy (cases), whereas 91 had no evidence of the disease (controls). The VEGF (-2578C>A, -460T>C and +936C>T) and KDR (-604C>T, 1192G>A and 1719T>A) polymorphisms were determined by TaqMan real-time polymerase chain reaction. The odds ratio (OR) with their 95% confidence intervals (CI) were calculated using an unconditional logistic regression model. The evaluation of demographic and clinical characteristics revealed the athlete age (P volleyball (P T, 1192G>A and 1719T>A) haplotypes CGA and CAT were associated with decreased tendinopathy risk (OR: 0.46, 95% CI: 0.21-0.99 and OR: 0.23, 95% CI: 0.07-0.76, respectively). With regards to pain, traumatic lesion and away from training due to injury, VEGF and KDR polymorphisms were not associated with clinical symptoms complaints. The present results provide evidence that the KDR polymorphisms were associated with development of tendinopathy, and can contribute to identify new therapeutic targets or personalized training programs to avoid tendinopathy development in athletes.

  20. Congenital CMV infection: prevalence in newborns and the impact on hearing deficit.

    Science.gov (United States)

    Engman, Mona-Lisa; Malm, Gunilla; Engstrom, Lotta; Petersson, Karin; Karltorp, Eva; Tear Fahnehjelm, Kristina; Uhlen, Inger; Guthenberg, Claes; Lewensohn-Fuchs, Ilona

    2008-01-01

    Congenital cytomegalovirus (CMV) infection is asymptomatic in 90% of infected newborns but approximately 10-20% of these infants are at risk of developing sequelae later, mostly hearing deficit. The aims of the study were to investigate the prevalence of congenital CMV infection in a Swedish population of newborns and investigate the relative risk of hearing deficit in newborns with congenital CMV infection. The dried blood spot (DBS) samples of 6060 newborns in southern Stockholm during 12 months (October 2003-June 2004; August 2004-October 2004) were analysed for CMV DNA by TaqMan based real-time PCR. Hearing deficit was assessed by otoacoustic emission (OAE) within a newborn screening programme. 12 infants out of 6060 or 0.2% (95% CI 0.1-0.3%) had congenital CMV infection. One boy among the 12 infected infants had unilateral hearing loss, indicating that the risk of hearing loss is greatly increased (about 20 times) in CMV infected infants. No child developed ocular complications such as chorioretinopathy during 3 y of follow-up. Congenital CMV has an impact on child health but can easily be overlooked due to lack of signs in the neonatal period. Surveillance for congenital CMV is important in addition to programmes for prevention and treatment.

  1. The prognostic value of p53 mutation in pediatric marrow hypoplasia

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    Sharaf Alzahraa EA

    2011-06-01

    Full Text Available Abstract Background The tumor suppressor gene p53 is involved in the control of cell proliferation, particularly in stressed cells. p 53 gene mutations are the most frequent genetic event found in human cancers. Fanconi Anemia (FA is the most common representative of inherited bone marrow failure syndromes (IBMFS with a leukemic propensity. P 53 DNA alteration has not been studied before in Egyptian children with FA. Patients and methods we investigated p53 mutation in the bone marrow and peripheral blood of forty children, FA (n = 10, acquired aplastic anemia (AAA (n = 10, and immune thrombocytopenia (ITP as a control (n = 20, using real-time PCR by TaqMan probe assay Results Mutation of p53 gene was demonstrated in the BM of 90% (9/10 of children with FA, compared to 10% (1/10 in AAA (p Conclusion mutation of p53 gene in hypoplastic marrow especially FA may represent an early indicator of significant DNA genetic alteration with cancer propensity.

  2. Genome-wide copy number variation (CNV in patients with autoimmune Addison's disease

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    Brønstad Ingeborg

    2011-08-01

    Full Text Available Abstract Background Addison's disease (AD is caused by an autoimmune destruction of the adrenal cortex. The pathogenesis is multi-factorial, involving genetic components and hitherto unknown environmental factors. The aim of the present study was to investigate if gene dosage in the form of copy number variation (CNV could add to the repertoire of genetic susceptibility to autoimmune AD. Methods A genome-wide study using the Affymetrix GeneChip® Genome-Wide Human SNP Array 6.0 was conducted in 26 patients with AD. CNVs in selected genes were further investigated in a larger material of patients with autoimmune AD (n = 352 and healthy controls (n = 353 by duplex Taqman real-time polymerase chain reaction assays. Results We found that low copy number of UGT2B28 was significantly more frequent in AD patients compared to controls; conversely high copy number of ADAM3A was associated with AD. Conclusions We have identified two novel CNV associations to ADAM3A and UGT2B28 in AD. The mechanism by which this susceptibility is conferred is at present unclear, but may involve steroid inactivation (UGT2B28 and T cell maturation (ADAM3A. Characterization of these proteins may unravel novel information on the pathogenesis of autoimmunity.

  3. The PAI-1 4G/5G and ACE I/D polymorphisms and risk of recurrent pregnancy loss: a case-control study.

    Science.gov (United States)

    Kim, Jin Ju; Choi, Young Min; Lee, Sung Ki; Yang, Kwang Moon; Paik, Eun Chan; Jeong, Hyeon Jeong; Jun, Jong Kwan; Han, Ae Ra; Hong, Min A

    2014-12-01

    Thrombophilia has been postulated to be a contributor to the pathophysiology of recurrent pregnancy loss (RPL). We investigated the role of the plasminogen activator inhibitor type 1 (PAI-1) 4G/5G and angiotensin converting enzyme (ACE) I/D polymorphisms in Korean patients with RPL. Genotyping was performed using the TaqMan assay in 227 RPL patients and 304 controls. The genotype distributions of both polymorphisms in the RPL group did not differ from those of controls. Because the frequency of being homozygous for ACE D/D and the PAI-I 4G/4G combination has been reported to be significantly higher in RPL patients, this was also analyzed. However, no significant difference was noted; 3.1% of RPL patients had both ACE D/D and PAI-I 4G/4G, as did 4.9% of controls (P = 0.791). The current study suggests that both polymorphisms, either alone or in combination, are not major determinants of the development of RPL in Korean women. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Infectious mononucleosis-linked HLA class I single nucleotide polymorphism is associated with multiple sclerosis.

    Science.gov (United States)

    Jafari, Naghmeh; Broer, Linda; Hoppenbrouwers, Ilse A; van Duijn, Cornelia M; Hintzen, Rogier Q

    2010-11-01

    Multiple sclerosis is a presumed autoimmune disease associated with genetic and environmental risk factors such as infectious mononucleosis. Recent research has shown infectious mononucleosis to be associated with a specific HLA class I polymorphism. Our aim was to test if the infectious mononucleosis-linked HLA class I single nucleotide polymorphism (rs6457110) is also associated with multiple sclerosis. Genotyping of the HLA-A single nucleotide polymorphism rs6457110 using TaqMan was performed in 591 multiple sclerosis cases and 600 controls. The association of multiple sclerosis with the HLA-A single nucleotide polymorphism was tested using logistic regression adjusted for age, sex and HLA-DRB1*1501. HLA-A minor allele (A) is associated with multiple sclerosis (OR = 0.68; p = 4.08 × 10( -5)). After stratification for HLA-DRB1*1501 risk allele (T) carrier we showed a significant OR of 0.70 (p = 0.003) for HLA-A. HLA class I single nucleotide polymorphism rs6457110 is associated with infectious mononucleosis and multiple sclerosis, independent of the major class II allele, supporting the hypothesis that shared genetics may contribute to the association between infectious mononucleosis and multiple sclerosis.

  5. Valacyclovir Pharmacokinetics and Exploratory Pharmacodynamics in Young Adults With Epstein-Barr Virus Infectious Mononucleosis

    Science.gov (United States)

    Vezina, Heather E.; Balfour, Henry H.; Weller, Dennis R.; Anderson, Bruce J.; Brundage, Richard C.

    2017-01-01

    Primary Epstein-Barr virus (EBV) infection often results in infectious mononucleosis and is associated with serious sequelae. No treatment is approved for EBV infection, and an antiviral intervention would be significant. The objectives of this study are to characterize the pharmacokinetics and explore the pharmacodynamics of acyclovir in plasma and oral washings of 8 subjects receiving 7 days of valacyclovir 1500 mg twice daily for EBV infectious mononucleosis. Virologic and clinical responses are assessed over 12 days. Acyclovir is measured by liquid chromatography/ultraviolet detection. EBV DNA is quantitated by TaqMan polymerase chain reaction. NONMEM VI and linear regression are used for data analysis. Acyclovir profiles in plasma and oral washings are consistent with a 1-compartment model. Final model estimates of clearance, volume of distribution, and fraction of acyclovir in oral wash supernatant are 49.9 L/h, 74.1 L, and 1.14%, respectively. The quantity of EBV DNA in oral washings and blood, and the severity of illness, measured by a graded scale, decrease during treatment. After treatment, viral rebound occurs in oral washings but not in blood, and the severity of illness continues to decline. Acyclovir pharmacokinetic parameters do not correlate with response metrics. These results support further studies of valacyclovir for EBV infectious mononucleosis. PMID:19897764

  6. Piwil1 mediates meiosis during spermatogenesis in chicken.

    Science.gov (United States)

    Xu, Lu; Chang, Guobin; Ma, Teng; Wang, Hongzhi; Chen, Jing; Li, Zhiteng; Guo, Xiaomin; Wan, Fang; Ren, Lichen; Lu, Wei; Chen, Guohong

    2016-03-01

    Piwil1 mediates spermatogenesis and ensures stable cell division rates in germline cells in mammals. However, the involvement of Piwil1 in poultry spermatogenesis and meiosis is poorly understood. In the present study, we used TaqMan RT-qPCR to characterize Piwil1 mRNA expression in different types of spermatogenic cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), spermatogonia cells (Sa), tetraploid cells (Tp), round sperm cells (Rs), mature sperm, and in PGCs treated with retinoic acid. Our results revealed that Piwil1 is differentially expressed during spermatogenesis in chicken. Compared to PGCs, SSCs, Tp, and Sa, Rs cells presented the highest Piwil1 mRNA expression levels. Retinoic acid significantly upregulated Piwil1 and Stra8 mRNA expression as well as Piwil1 levels in chicken PGCs. In addition, retinoic acid induced PGCs to progress through all the meiotic stages, eventually leading to haploid cell formation, which was determined using flow cytometry and western blot analysis. Taken together, our results showed that during spermatogenesis, Piwil1 was first expressed at low levels in germ stem cells, PGCs, and SSCs. Its expression levels increased during later meiosis stages. Finally, no expression was detected in mature sperm after meiosis. Treatment of PGCs with retinoic acid further demonstrated that Piwil1 plays a key role in meiosis during chicken spermatogenesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Let7a involves in neural stem cell differentiation relating with TLX level.

    Science.gov (United States)

    Song, Juhyun; Cho, Kyoung Joo; Oh, Yumi; Lee, Jong Eun

    2015-07-10

    Neural stem cells (NSCs) have the potential for differentiation into neurons known as a groundbreaking therapeutic solution for central nervous system (CNS) diseases. To resolve the therapeutic efficiency of NSCs, recent researchers have focused on the study on microRNA's role in CNS. Some micro RNAs have been reported significant functions in NSC self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. MicroRNA-Let7a (Let7a) has known as the regulator of diverse cellular mechanisms including cell differentiation and proliferation. In present study, we investigated whether Let7a regulates NSC differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal, proliferation and differentiation. We performed the following experiments: western blot analysis, TaqMan assay, RT-PCR, and immunocytochemistry to confirm the alteration of NSCs. Our data showed that let7a play important roles in controlling NSC fate determination. Thus, manipulating Let-7A and TLX could be a novel strategy to enhance the efficiency of NSC's neuronal differentiation for CNS disorders. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Molecular Endoscopic Ultrasound for Diagnosis of Pancreatic Cancer

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    Bournet, Barbara [Department of Gastroenterology, University Hospital Center Rangueil, 1 avenue Jean Poulhès, TSA 50032, 31059 Toulouse Cedex 9 (France); INSERM U1037, University Hospital Center Rangueil, Toulouse (France); Pointreau, Adeline; Delpu, Yannick; Selves, Janick; Torrisani, Jerome [INSERM U1037, University Hospital Center Rangueil, Toulouse (France); Buscail, Louis, E-mail: buscail.l@chu-toulouse.fr [Department of Gastroenterology, University Hospital Center Rangueil, 1 avenue Jean Poulhès, TSA 50032, 31059 Toulouse Cedex 9 (France); INSERM U1037, University Hospital Center Rangueil, Toulouse (France); Cordelier, Pierre [INSERM U1037, University Hospital Center Rangueil, Toulouse (France)

    2011-02-24

    Endoscopic ultrasound-guided fine needle aspiration-biopsy is a safe and effective technique in diagnosing and staging of pancreatic ductal adenocarcinoma. However its predictive negative value does not exceed 50% to 60%. Unfortunately, the majority of pancreatic cancer patients have a metastatic and/or a locally advanced disease (i.e., not eligible for curative resection) which explains the limited access to pancreatic tissue specimens. Endoscopic ultrasound-guided fine needle aspiration-biopsy is the most widely used approach for cytological and histological material sampling in these situations used in up to two thirds of patients with pancreatic cancer. Based on this unique material, we and others developed strategies to improve the differential diagnosis between carcinoma and inflammatory pancreatic lesions by analysis of KRAS oncogene mutation, microRNA expression and methylation, as well as mRNA expression using both qRT-PCR and Low Density Array Taqman analysis. Indeed, differentiating pancreatic cancer from pseudotumoral chronic pancreatitis remains very difficult in current clinical practice, and endoscopic ultrasound-guided fine needle aspiration-biopsy analysis proved to be very helpful. In this review, we will compile the clinical and molecular advantages of using endoscopic ultrasound-guided fine needle aspiration-biopsy in managing pancreatic cancer.

  9. [Characterisation of three polymorphisms of the tryptophan hydroxylase 2 gene in a sample of Colombian population with major depressive disorder].

    Science.gov (United States)

    Martínez-Idárraga, Adriana; Riveros-Barrera, Irene; Sánchez, Ricardo; Jaramillo, Luis Eduardo; Calvo-Gómez, José Manuel; Yunis-Londoño, Juan José

    Identify whether rs11179000, rs136494 and rs4570625 polymorphisms of the tryptophan hydroxylase 2 gene, are associated with a major depressive disorder in a sample of the Colombian population. Case-control study was conducted in which a comparison was made between subjects diagnosed with major depressive disorder at some point in adulthood or active symptoms at the time of evaluation, and subjects with no psychiatric disease. Subjects were studied in the Department of Psychiatry, Faculty of Medicine and the Institute of Genetics at the National University of Colombia. Polymorphisms were genotyped using Taqman probes in real time PCR. As well as studying the association between major depressive disorder and these (single nucleotide polymorphisms (SNPs), the association with other factors previously associated with depression were also analysed. No statistically significant association between genotypic and allelic frequencies of each polymorphism and major depressive disorder was found. Association between sex and complication during pregnancy / childbirth and major depressive disorder was observed. Association between sex and complication during pregnancy / childbirth and major depressive disorder was observed. There was no association between any polymorphism and major depressive disorder. Copyright © 2016 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

  10. Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials.

    Science.gov (United States)

    Soejima, Mikiko; Tsuchiya, Yuji; Egashira, Kouichi; Kawano, Hiroyuki; Sagawa, Kimitaka; Koda, Yoshiro

    2010-06-01

    Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

  11. A novel duplex real-time reverse transcriptase-polymerase chain reaction assay for the detection of hepatitis C viral RNA with armored RNA as internal control

    Directory of Open Access Journals (Sweden)

    Meng Shuang

    2010-06-01

    Full Text Available Abstract Background The hepatitis C virus (HCV genome is extremely heterogeneous. Several HCV infections can not be detected using currently available commercial assays, probably because of mismatches between the template and primers/probes. By aligning the HCV sequences, we developed a duplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR assay using 2 sets of primers/probes and a specific armored RNA as internal control. The 2 detection probes were labelled with the same fluorophore, namely, 6-carboxyfluorescein (FAM, at the 5' end; these probes could mutually combine, improving the power of the test. Results The limit of detection of the duplex primer/probe assay was 38.99 IU/ml. The sensitivity of the assay improved significantly, while the specificity was not affected. All HCV genotypes in the HCV RNA Genotype Panel for Nucleic Acid Amplification Techniques could be detected. In the testing of 109 serum samples, the performance of the duplex real-time RT-PCR assay was identical to that of the COBAS AmpliPrep (CAP/COBAS TaqMan (CTM assay and superior to 2 commercial HCV assay kits. Conclusions The duplex real-time RT-PCR assay is an efficient and effective viral assay. It is comparable with the CAP/CTM assay with regard to the power of the test and is appropriate for blood-donor screening and laboratory diagnosis of HCV infection.

  12. Entomologic investigations during an outbreak of West Nile virus disease in Maricopa County, Arizona, 2010.

    Science.gov (United States)

    Godsey, Marvin S; Burkhalter, Kristen; Young, Ginger; Delorey, Mark; Smith, Kirk; Townsend, John; Levy, Craig; Mutebi, John-Paul

    2012-12-01

    Entomologic investigations were conducted during an intense outbreak of West Nile virus (WNV) disease in Maricopa County, Arizona during July 31-August 9, 2010. The investigations compared the East Valley outbreak area, and a demographically similar control area in northwestern metropolitan Phoenix where no human cases were reported. Five mosquito species were identified in each area, and species composition was similar in both areas. Significantly more Culex quinquefasciatus females were collected by gravid traps at Outbreak sites (22.2 per trap night) than at control sites (8.9 per trap night), indicating higher Cx. quinquefasciatus abundance in the outbreak area. Twenty-eight WNV TaqMan reverse transcription-polymerase chain reaction-positive mosquito pools were identified, including 24 of Cx. quinquefasciatus, 3 of Psorophora columbiae, and 1 of Culex sp. However, Cx. quinquefasciatus WNV infection rates did not differ between outbreak and control sites. At outbreak sites, 30 of 39 engorged Cx. quinquefasciatus had fed on birds, 8 of 39 on humans, and 1 of 39 on a lizard. At control sites, 20 of 20 identified blood meals were from birds. Data suggest that Cx. quinquefasciatus was the primary enzootic and epidemic vector of this outbreak. The most important parameters in the outbreak were vector abundance and blood meal analysis, which suggested more frequent contact between Cx. quinquefasciatus and human hosts in the outbreak area compared with the control area.

  13. Differences in UGT1A1, UGT1A7, and UGT1A9 polymorphisms between Uzbek and Japanese populations.

    Science.gov (United States)

    Maeda, Hiromichi; Hazama, Shoichi; Shavkat, Abdiev; Okamoto, Ken; Oba, Koji; Sakamoto, Junichi; Takahashi, Kenichi; Oka, Masaki; Nakamura, Daisuke; Tsunedomi, Ryouichi; Okayama, Naoko; Mishima, Hideyuki; Kobayashi, Michiya

    2014-06-01

    Uridine-diphosphate glucuronosyltransferase 1A (UGT1A) is a key enzyme involved in irinotecan metabolism, and polymorphisms in the UGT1A gene are associated with irinotecan-induced toxicity. The aim of this study was to elucidate the allele frequencies of UGT1A polymorphisms in healthy Uzbek volunteers, and to compare them with those of the Japanese population. A total of 97 healthy volunteers from Uzbekistan were enrolled and blood samples were collected from each participant. Genotyping analysis was performed by fragment size analysis for UGT1A1*28, direct sequencing for UGT1A7*3 and UGT1A9*22, and TaqMan assays for UGT1A1*93, UGT1A1*6, UGT1A1*27, UGT1A1*60, and UGT1A7*12. The frequencies of polymorphisms were compared with the Japanese population by using the data previously reported from our study group. When the Uzbek and Japanese populations were compared, heterozygotes or homozygotes for UGT1A1*28, UGT1A1*60, and UGT1A1*93 were significantly more frequent in the Uzbek population (P Japanese population (P Japanese population. A comprehensive study of the influence of UGT1A1 polymorphisms on the risk of irinotecan-induced toxicity is necessary for optimal use of irinotecan treatment.

  14. IRF5, PTPN22, CD28, IL2RA, KIF5A, BLK and TNFAIP3 genes polymorphisms and lupus susceptibility in a cohort from the Egypt Delta; relation to other ethnic groups.

    Science.gov (United States)

    Elghzaly, Ashraf A; Metwally, Shereen S; El-Chennawi, Farha A; Elgayaar, Maha A; Mosaad, Youssef M; El-Toraby, Ehab E; Hegab, Mohsen M; Ibrahim, Saleh M

    2015-07-01

    To replicate a single nucleotide polymorphism (SNP) of known genes for lupus (IRF5 rs10488631, PTPN22 rs2476601, BLK rs2736340 and TNFAIP3 rs5029939) and other autoimmune diseases (CD28 rs1980422, IL2RA rs2104286 and KIF5A rs1678542) on a newly studied Egyptian cohort to investigate the genetic disparity with different studied ethnic groups in relation to lupus susceptibility. 170 Egyptian patients from Egypt Delta with SLE and 241 matched healthy controls were genotyped by Taqman real time PCR for the selected SNPs. The results revealed significant association with IRF5 (p<0.0001) and PTPN22 (p=0.008) and insignificant association with KIF5A, CD28, IL2RA, BLK and TNFAIP3 genes. This study may provide an additional evidence for the association between IRF5 and PTPN22 and lupus susceptibility and may exclude it for CD28, IL2RA, and KIF5A. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  15. Molecular diagnostics for the sigatoka disease complex of banana.

    Science.gov (United States)

    Arzanlou, Mahdi; Abeln, Edwin C A; Kema, Gert H J; Waalwijk, Cees; Carlier, Jean; Vries, Ineke de; Guzmán, Mauricio; Crous, Pedro W

    2007-09-01

    ABSTRACT The Sigatoka disease complex of banana involves three related ascomycetous fungi, Mycosphaerella fijiensis, M. musicola, and M. eumusae. The exact distribution of these three species and their disease epidemiology remain unclear, because their symptoms and life cycles are rather similar. Disease diagnosis in the Mycosphaerella complex of banana is based on the presence of host symptoms and fungal fruiting structures, which hamper preventive management strategies. In the present study, we have developed rapid and robust species-specific molecular-based diagnostic tools for detection and quantification of M. fijiensis, M. musicola, and M. eumusae. Conventional species-specific polymerase chain reaction (PCR) primers were developed based on the actin gene that detected DNA at as little as 100, 1, and 10 pg/mul from M. fijiensis, M. musicola, and M. eumusae, respectively. Furthermore, TaqMan real-time quantitative PCR assays were developed based on the beta-tubulin gene and detected quantities of DNA as low as 1 pg/mul for each Mycosphaerella sp. from pure cultures and DNA at 1.6 pg/mul per milligram of dry leaf tissue for M. fijiensis that was validated using naturally infected banana leaves.

  16. Changes in human fecal microbiota due to chemotherapy analyzed by TaqMan-PCR, 454 sequencing and PCR-DGGE fingerprinting.

    Directory of Open Access Journals (Sweden)

    Jutta Zwielehner

    Full Text Available BACKGROUND: We investigated whether chemotherapy with the presence or absence of antibiotics against different kinds of cancer changed the gastrointestinal microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Feces of 17 ambulant patients receiving chemotherapy with or without concomitant antibiotics were analyzed before and after the chemotherapy cycle at four time points in comparison to 17 gender-, age- and lifestyle-matched healthy controls. We targeted 16S rRNA genes of all bacteria, Bacteroides, bifidobacteria, Clostridium cluster IV and XIVa as well as C. difficile with TaqMan qPCR, denaturing gradient gel electrophoresis (DGGE fingerprinting and high-throughput sequencing. After a significant drop in the abundance of microbiota (p = 0.037 following a single treatment the microbiota recovered within a few days. The chemotherapeutical treatment marginally affected the Bacteroides while the Clostridium cluster IV and XIVa were significantly more sensitive to chemotherapy and antibiotic treatment. DGGE fingerprinting showed decreased diversity of Clostridium cluster IV and XIVa in response to chemotherapy with cluster IV diversity being particularly affected by antibiotics. The occurrence of C. difficile in three out of seventeen subjects was accompanied by a decrease in the genera Bifidobacterium, Lactobacillus, Veillonella and Faecalibacterium prausnitzii. Enterococcus faecium increased following chemotherapy. CONCLUSIONS/SIGNIFICANCE: Despite high individual variations, these results suggest that the observed changes in the human gut microbiota may favor colonization with C. difficile and Enterococcus faecium. Perturbed microbiota may be a target for specific mitigation with safe pre- and probiotics.

  17. Low-Dose Fluvastatin and Valsartan Rejuvenate the Arterial Wall Through Telomerase Activity Increase in Middle-Aged Men.

    Science.gov (United States)

    Janić, Miodrag; Lunder, Mojca; Cerkovnik, Petra; Prosenc Zmrzljak, Uršula; Novaković, Srdjan; Šabovič, Mišo

    2016-04-01

    Previously, we have shown that slightly to moderately aged arteries in middle-aged males can be rejuvenated functionally by sub-therapeutic, low-dose fluvastatin and valsartan treatment. Here, we explore whether this treatment could also increase telomerase activity. We hypothesized that telomerase activity might be associated with (1) an improvement of arterial wall properties and (2) a reduction of inflammatory/oxidative stress parameters (both observed in our previous studies). The stored blood samples from 130 apparently healthy middle-aged males treated with fluvastatin (10 mg daily), valsartan (20 mg daily), fluvastatin and valsartan combination (10 and 20 mg), respectively, and placebo (control), were analyzed. The samples were taken before and after treatment lasting 30 days, and 5 months after treatment discontinuation. Telomerase activity was measured in blood leukocytes by a TaqMan Gene Expression Assay. Low-dose fluvastatin or valsartan increased telomerase activity (106.9% and 59.5% respectively; both p valsartan substantially increased telomerase activity, which significantly correlated with an improvement of endothelial function and a decrease of inflammation/oxidative stress. These findings could lead to a new innovative approach to arterial rejuvenation.

  18. Genome-wide copy number variation (CNV) in patients with autoimmune Addison's disease

    Science.gov (United States)

    2011-01-01

    Background Addison's disease (AD) is caused by an autoimmune destruction of the adrenal cortex. The pathogenesis is multi-factorial, involving genetic components and hitherto unknown environmental factors. The aim of the present study was to investigate if gene dosage in the form of copy number variation (CNV) could add to the repertoire of genetic susceptibility to autoimmune AD. Methods A genome-wide study using the Affymetrix GeneChip® Genome-Wide Human SNP Array 6.0 was conducted in 26 patients with AD. CNVs in selected genes were further investigated in a larger material of patients with autoimmune AD (n = 352) and healthy controls (n = 353) by duplex Taqman real-time polymerase chain reaction assays. Results We found that low copy number of UGT2B28 was significantly more frequent in AD patients compared to controls; conversely high copy number of ADAM3A was associated with AD. Conclusions We have identified two novel CNV associations to ADAM3A and UGT2B28 in AD. The mechanism by which this susceptibility is conferred is at present unclear, but may involve steroid inactivation (UGT2B28) and T cell maturation (ADAM3A). Characterization of these proteins may unravel novel information on the pathogenesis of autoimmunity. PMID:21851588

  19. Nine year trends of dengue virus infection in Mumbai, Western India

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    Jayanthi Shastri

    2017-01-01

    Methods and Results: During the nine year period of this study analysis, 6767 strongly suspected cases were tested by RT-PCR. 1685 (24.9% were Dengue PCR positive and confirmed as Dengue cases. Observations on the seasonality were based on the nine year's data as the intensity of sampling was at its maximum during monsoon season. Dengue typing was done on 100 positive samples after storage of Dengue RNA at – 80°C. Dengue serotypes were detected in 69 samples of which Dengue 2 was most predominant. 576 samples were processed for NS1 antigen and PCR simultaneously. 19/576 were positive (3.3 % for NS1 as well as by PCR . 23/576 samples were negative for NS1 antigen, but were positive by RT-PCR. The remaining 534 samples which were negative for NS1 antigen were also negative by Dengue RT-PCR. Conclusion: In this study we sought to standardize rapid, sensitive, and specific fluorogenic probe-based RT-PCR assay to screen and serotype a representative range of Dengue viruses that are found in and around Mumbai. Qualitative Dengue virus TaqMan assays could have tremendous utility for the epidemiological investigation of Dengue illness and especially for the study of the viremic response with candidate live-attenuated dengue virus vaccines.

  20. Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery.

    Science.gov (United States)

    Schnor, Noa Pereira Prada; Verlengia, Rozangela; Novais, Patrícia Fátima Sousa; Crisp, Alex Harley; Leite, Celso Vieira de Souza; Rasera-Junior, Irineu; Oliveira, Maria Rita Marques de

    2017-01-01

    Obesity can cause systemic arterial hypertension (SAH) and type 2 diabetes mellitus (DM2) factor that is also influenced by genetic variability. The present study aims to investigate the association between gene polymorphisms related with obesity on the prevalence of SAH and DM2 in the preoperative period and 1 year after Roux-en-Y gastric bypass surgery. In total, 351 obese women in a Brazilian cohort completed the study. The clinical diagnosis of SAH and DM2 was monitored from medical records. Twelve gene polymorphisms (rs26802; rs572169; rs7799039; rs1137101; rs3813929; rs659366; rs660339; rs1800849; rs7498665; rs35874116; rs9701796; and rs9939609) were determined using real-time polymerase chain reaction and TaqMan assay. In the preoperative period, prevalence of SAH and DM2 was 57% and 22%, respectively. One year postoperatively, 86.8% subjects had remission of DM2 and 99.5% had control of SAH. Subjects with T allele from the serotonin receptor gene (5-HT2C, rs3813929) had five times greater chance of DM2, and the CC genotype from uncoupling protein 3 gene (UCP3, rs1800849) had three times greater chance in the preoperative period. These findings indicate that polymorphisms rs3813929 and rs1800849 from 5-HT2C and UCP3 genes were related to DM2 prevalence among the Brazilian obese women candidates for bariatric surgery.

  1. Identification of several circulating microRNAs from a genome-wide circulating microRNA expression profile as potential biomarkers for impaired glucose metabolism in polycystic ovarian syndrome.

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    Jiang, Linlin; Huang, Jia; Chen, Yaxiao; Yang, Yabo; Li, Ruiqi; Li, Yu; Chen, Xiaoli; Yang, Dongzi

    2016-07-01

    This study aimed to detect serum microRNAs (miRNAs) differentially expressed between polycystic ovary syndrome (PCOS) patients with impaired glucose metabolism (IGM), PCOS patients with normal glucose tolerance (NGT), and healthy controls. A TaqMan miRNA array explored serum miRNA profiles as a pilot study, then selected miRNAs were analyzed in a validation cohort consisting of 65 PCOS women with IGM, 65 PCOS women with NGT, and 45 healthy women The relative expression of miR-122, miR-193b, and miR-194 was up-regulated in PCOS patients compared with controls, whereas that of miR-199b-5p was down-regulated. Furthermore, miR-122, miR-193b, and miR-194 were increased in the PCOS-IGM group compared with the PCOS-NGT group. Multiple linear regression analyses revealed that miR-193b and body mass index contributed independently to explain 43.7 % (P ovarian follicle development pathways, including the insulin signaling pathway, the neurotrophin signaling pathway, the PI3K-AKT signaling pathway, and regulation of actin cytoskeleton. This study expands our knowledge of the serum miRNA expression profiles of PCOS patients with IGM and the predicted target signal pathways involved in disease pathophysiology.

  2. Role of serum miRNAs in the prediction of ovarian hyperstimulation syndrome in polycystic ovarian syndrome patients.

    Science.gov (United States)

    Zhao, Chun; Liu, Xiaoguang; Shi, Zhonghua; Zhang, Jing; Zhang, Junqiang; Jia, Xuemei; Ling, Xiufeng

    2015-01-01

    Polycystic ovarian syndrome (PCOS) causes a significantly increased risk of ovarian hyperstimulation syndrome (OHSS). Here, we focused on the altered expression of serum miRNAs and their predictive value for OHSS in PCOS patients. We used the TaqMan low density array followed by individual quantitative reverse transcription-polymerase chain reaction to identify and validate the expression of serum miRNAs in PCOS patients likely to develop severe OHSS. The miR-16 and miR-223 expression levels were significantly reduced in the patients who were likely to develop severe OHSS than in the control subjects who were likely to develop mild or no OHSS. The sensitivity and specificity of the basal LH, basal LH/FSH, and body mass index (BMI) as OHSS predictors were also evaluated. miR-16 was the most efficient for OHSS prediction as it yielded the highest AUC. Logistic binary regression analyses revealed a positive association of miR-223 and BMI. Serum miRNAs are differentially expressed in PCOS patients likely to suffer from severe OHSS. We identified and validated two serum miRNAs that have potential for use as novel noninvasive biomarkers to accurately predict OHSS before controlled ovarian hyperstimulation (COH) for PCOS patients. © 2015 S. Karger AG, Basel.

  3. Real-Time Polymerase Chain Reaction Detection of Trichomonas vaginalis from vaginal swabs: Validation of a Diagnostic Method and Preliminary Epidemiological Application

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    Carlo Mengoli

    2009-06-01

    Full Text Available Background Trichomonas vaginalis is the most common nonviral sexually trasmitted diseases (STDs agent. For females, the diagnostic gold standard is the culture of vaginal swab, which is labour-exacting.The direct microscopic examination of vaginal secretions is the most used approach, but its sensitivity depends on the skill of the observer. Objectives We evaluated an original real-time TaqMan-based Polymerase Chain Reaction (PCR technique.The scope of the study was to confirm the effectiveness of the molecular approach in a clinical context and to explore its relevance to an epidemiological investigation. Study Design a ß-tubulin gene was chosen as target sequence.The assay was designed to exploit the quantitative potential of the TaqMan procedure.The population sample was 583 adult females presenting at the Service from January 2005 to December 2005.Three vaginal swabs were collected from each patient, one for wet mount microscopy, one for broth culture, and one for the molecular assay. Results The prevalence was 3.3% (culture, 3.1% (microscopy, 3.8% (PCR.An excess risk was detected in the immigrant population (risk ratio by PCR = 28. Conclusions The molecular approach was the most accurate way to detect the protozoon.The real-time PCR is convenient in a busy laboratory, provided the necessary equipment is available, and it is suitable for epidemiological investigation.

  4. Different contribution of BRINP3 gene in chronic periodontitis and peri-implantitis: a cross-sectional study.

    Science.gov (United States)

    Casado, Priscila L; Aguiar, Diego P; Costa, Lucas C; Fonseca, Marcos A; Vieira, Thays C S; Alvim-Pereira, Claudia C K; Alvim-Pereira, Fabiano; Deeley, Kathleen; Granjeiro, José M; Trevilatto, Paula C; Vieira, Alexandre R

    2015-03-11

    Peri-implantitis is a chronic inflammation, resulting in loss of supporting bone around implants. Chronic periodontitis is a risk indicator for implant failure. Both diseases have a common etiology regarding inflammatory destructive response. BRINP3 gene is associated with aggressive periodontitis. However, is still unclear if chronic periodontitis and peri-implantitis have the same genetic background. The aim of this work was to investigate the association between BRINP3 genetic variation (rs1342913 and rs1935881) and expression and susceptibility to both diseases. Periodontal and peri-implant examinations were performed in 215 subjects, divided into: healthy (without chronic periodontitis and peri-implantitis, n = 93); diseased (with chronic periodontitis and peri-implantitis, n = 52); chronic periodontitis only (n = 36), and peri-implantitis only (n = 34). A replication sample of 92 subjects who lost implants and 185 subjects successfully treated with implants were tested. DNA was extracted from buccal cells. Two genetic markers of BRINP3 (rs1342913 and rs1935881) were genotyped using TaqMan chemistry. Chi-square (p chronic periodontitis.

  5. Microbiological Quality of Fresh Produce from Open Air Markets and Supermarkets in the Philippines

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    Pierangeli G. Vital

    2014-01-01

    Full Text Available This study is the first in the Philippines to conduct a comprehensive assessment of the prevalence of bacterial pathogens and somatic phages in retailed fresh produce used in salad preparation, namely, bell pepper, cabbage, carrot, lettuce, and tomato, using culture and molecular methods. Out of 300 samples from open air and supermarkets, 16.7% tested positive for thermotolerant Escherichia coli, 24.7% for Salmonella spp., and 47% for somatic phages. Results show that counts range from 0.30 to 4.03 log10 CFU/g for E. coli, 0.66 to ≥2.34 log10 MPN/g for Salmonella spp., and 1.30 to ≥3.00 log10 PFU/g for somatic phages. Statistical analyses show that there was no significant difference in the microbial counts between open air and supermarkets (α=0.05. TaqMan and AccuPower Plus DualStar real-time polymerase chain reaction (RT-PCR was used to confirm the presence of these organisms. The relatively high prevalence of microorganisms observed in produce surveyed signifies reduction in shelf-life and a potential hazard to food safety. This information may benefit farmers, consumers, merchants, and policy makers for foodborne disease detection and prevention.

  6. Interaction between β-hexachlorocyclohexane and ADIPOQ genotypes contributes to the risk of type 2 diabetes mellitus in East Chinese adults

    Science.gov (United States)

    Li, Shushu; Wang, Xichen; Yang, Lu; Yao, Shen; Zhang, Ruyang; Xiao, Xue; Zhang, Zhan; Wang, Li; Xu, Qiujin; Wang, Shou-Lin

    2016-11-01

    Growing evidence links environmental exposure to hexachlorocyclohexanes (HCHs) to the risk of type 2 diabetes mellitus (T2DM), and ADIPOQ that encodes adiponectin is considered as an important gene for T2DM. However, the role of ADIPOQ-HCH interaction on T2DM risk remains unclear. Thus, a paired case-control study was conducted in an East Chinese community. A total of 1446 subjects, including 723 cases and 723 controls matched on age, gender and residence, were enrolled, and 4 types of HCH isomers were measured in serum samples using GC-MS/MS. Additionally, 4 candidate ADIPOQ SNPs (rs182052, rs266729, rs6810075, and rs16861194) were genotyped by TaqMan assay, and plasma adiponectin was measured using ELISA. No associations between 4 SNPs and T2DM risk were found, but T2DM risk significantly increased with serum levels of β-HCH (P 2052 significantly increased T2DM risk (OR I-additive model = 2.20, OR I-recessive model = 2.13). Additionally, individuals carrying only rs182052 (A allele) with high levels of β-HCH had significant reduction in adiponectin levels (P = 0.016). These results indicate that the interaction between rs182052 and β-HCH might increase the risk of T2DM by jointly decreasing the adiponectin level and potentially trigger T2DM development.

  7. Patient-specific induced pluripotent stem cells to evaluate the pathophysiology of TRNT1-associated Retinitis pigmentosa

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    Tasneem P. Sharma

    2017-05-01

    Full Text Available Retinitis pigmentosa (RP is a heterogeneous group of monogenic disorders characterized by progressive death of the light-sensing photoreceptor cells of the outer neural retina. We recently identified novel hypomorphic mutations in the tRNA Nucleotidyl Transferase, CCA-Adding 1 (TRNT1 gene that cause early-onset RP. To model this disease in vitro, we generated patient-specific iPSCs and iPSC-derived retinal organoids from dermal fibroblasts of patients with molecularly confirmed TRNT1-associated RP. Pluripotency was confirmed using rt-PCR, immunocytochemistry, and a TaqMan Scorecard Assay. Mutations in TRNT1 caused reduced levels of full-length TRNT1 protein and expression of a truncated smaller protein in both patient-specific iPSCs and iPSC-derived retinal organoids. Patient-specific iPSCs and iPSC-derived retinal organoids exhibited a deficit in autophagy, as evidenced by aberrant accumulation of LC3-II and elevated levels of oxidative stress. Autologous stem cell-based disease modeling will provide a platform for testing multiple avenues of treatment in patients suffering from TRNT1-associated RP.

  8. Evaluation of candidate genes associated with hepatitis A and E virus infection in Chinese Han population.

    Science.gov (United States)

    Gu, Maolin; Qiu, Jing; Guo, Daoxia; Xu, Yunfang; Liu, Xingxiang; Shen, Chong; Dong, Chen

    2018-03-20

    Recent GWAS-associated studies reported that single nucleotide polymorphisms (SNPs) in ABCB1, TGFβ1, XRCC1 genes were associated with hepatitis A virus (HAV) infection, and variants of APOA4 and APOE genes were associated with and hepatitis E virus (HEV) infection in US population. However, the associations of these loci with HAV or HEV infection in Chinese Han population remain unclear. A total of 3082 Chinese Han persons were included in this study. Anti-HAV IgG and anti-HEV IgG were detected by enzyme-linked immunosorbent assay (ELISA). Genotypes in ABCB1, TGFβ1, XRCC1, APOA4 and APOE SNPs were determined by TaqMan MGB technology. In Chinese Han population, rs1045642 C to T variation in ABCB1 was significantly associated with the decreased risk of HAV infection (P infection in our samples (P C to T variation in APOE was significantly associated with lower risk of HEV infection in males (adjusted OR infection. Additionally, Chinese Han males with rs7412 C to T variation in APOE gene are less prone to be infected by HEV.

  9. Additive effects of LPL, APOA5 and APOE variant combinations on triglyceride levels and hypertriglyceridemia: results of the ICARIA genetic sub-study

    Science.gov (United States)

    2010-01-01

    Background Hypertriglyceridemia (HTG) is a well-established independent risk factor for cardiovascular disease and the influence of several genetic variants in genes related with triglyceride (TG) metabolism has been described, including LPL, APOA5 and APOE. The combined analysis of these polymorphisms could produce clinically meaningful complementary information. Methods A subgroup of the ICARIA study comprising 1825 Spanish subjects (80% men, mean age 36 years) was genotyped for the LPL-HindIII (rs320), S447X (rs328), D9N (rs1801177) and N291S (rs268) polymorphisms, the APOA5-S19W (rs3135506) and -1131T/C (rs662799) variants, and the APOE polymorphism (rs429358; rs7412) using PCR and restriction analysis and TaqMan assays. We used regression analyses to examine their combined effects on TG levels (with the log-transformed variable) and the association of variant combinations with TG levels and hypertriglyceridemia (TG ≥ 1.69 mmol/L), including the covariates: gender, age, waist circumference, blood glucose, blood pressure, smoking and alcohol consumption. Results We found a significant lowering effect of the LPL-HindIII and S447X polymorphisms (p hypertriglyceridemia. PMID:20429872

  10. Interference of Homologous Sequences on the SNP Study of CYP2A13 Gene

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    Qinghua ZHOU

    2010-02-01

    Full Text Available Background and objective It has been proven that cytochrome P450 enzyme 2A13 (CYP2A13 played an important role in the association between single nucleotide polymorphisms (SNP and human diseases. Cytochrome P450 enzymes are a group of isoenzymes, whose sequence homology may interfere with the study for SNP. The aim of this study is to explore the interference on the SNP study of CYP2A13 caused by homologous sequences. Methods Taqman probe was applied to detect distribution of rs8192789 sites in 573 subjects, and BLAST method was used to analyze the amplified sequences. Partial sequences of CYP2A13 were emplified by PCR from 60 cases. The emplified sequences were TA cloned and sequenced. Results For rs8192789 loci in 573 cases, only 3 cases were TT, while the rest were CT heterozygotes, which was caused by homologous sequences. There are a large number of overlapping peaks in identical sequences of 60 cases, and the SNP of 101 amino acid site reported in the SNP database is not found. The cloned sequences are 247 bp, 235 bp fragments. Conclusion The homologous sequences may interfere the study for SNP of CYP2A13, and some SNP may not exist.

  11. Viral infection and oral habits as risk factors for oral squamous cell carcinoma in Yemen: a case-control study.

    Science.gov (United States)

    Nasher, Akram T; Al-Hebshi, Nezar N; Al-Moayad, Ebtisam E; Suleiman, Ahmed M

    2014-11-01

    The role of qat chewing, tobacco (shammah) dipping, smoking, alcohol drinking, and oral viral infection as risk factors for oral squamous cell carcinoma (OSCC) in Yemen was assessed. A total of 60 cases of OSCC and 120 age- and gender-matched controls were analyzed with respect to demographic data, history of oral habits, and the presence of human papillomavirus (HPV)-16, HPV-18, or Epstein-Barr virus (EBV) as determined by Taqman quantitative polymerase chain reaction. Logistic regression analysis was used to identify independent predictors of the disease. Shammah use was the only risk factor for OSCC, with an odds ratio of 12.6 (CI, 3.3-48.2) and 39 (CI, 14-105) for the ex-users and current users, respectively. The association of shammah use alone with OSCC exceeded that of shammah use in combination with qat chewing, smoking, or both. EBV infection, smoking, and qat chewing showed no association with OSCC, while neither HPV-16 nor HPV-18 were detected in any sample. Shammah use is a major risk factor for oral cancer in Yemen. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. PVR/CD155 Ala67Thr Mutation and Cleft Lip/Palate.

    Science.gov (United States)

    Vieira, Alexandre R; Letra, Ariadne; Silva, Renato M; Granjeiro, Jose M; Shimizu, Takehiko; Poletta, Fernando A; Mereb, Juan C; Castilla, Eduardo E; Orioli, Iêda M

    2018-03-01

    The 19q13 locus has been linked to cleft lip and palate by our group and independently by others. Here we fine mapped the region in an attempt to identify an etiological variant that can explain cleft lip and palate occurrence. A total of 2739 individuals born with cleft lip and palate, related to individuals born with cleft lip and palate, and unrelated were studied. We used linkage and association approaches to fine map the interval between D19S714 and D19S433 and genotypes were defined by the use of TaqMan chemistry. We confirmed our previous findings that markers in PVR/CD155 are associated with cleft lip and palate. We studied the mutation Ala67Thr further and calculated its penetrance. We also attempted to detect PVR/CD155 expression in human whole saliva. Our results showed that markers in PVR/CD155 are associated with cleft lip and palate and the penetrance of the Ala67Thr is very low (between 1% and 5%). We could not detect PVR/CD155 expression in adult human whole saliva and PVR/CD155 possibly interacts with maternal infection to predispose children to cleft lip only.

  13. The association of elastin gene variants with two angiographic subtypes of polypoidal choroidal vasculopathy.

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    Suiho Yanagisawa

    Full Text Available To compare the association of elastin (ELN gene variants between two different angiographic phenotypes of polypoidal choroidal vasculopathy (PCV.We included 411 treatment-naïve PCV patients and 350 controls in the present study. PCV was classified into two phenotypes (152 Type 1 and 259 Type 2 according to the presence or absence of feeding vessels found in indocyanine-green angiography. Single nucleotide polymorphisms (SNPs in the ELN region including rs868005, rs884843, rs2301995, rs13239907 and rs2856728 were genotyped using TaqMan Genotyping Assays.In the allelic association analyses, rs868005 showed the strongest association with Type 2 PCV (allelic odds ratio 1.56; p = 7.4x10(-6, while no SNP was significantly associated with Type 1 PCV. Genotype association analyses revealed the significant association of rs868005 with Type 2 PCV in log additive model and predominant model (odds ratio 1.75; p = 1.5x10(-6 and odds ratio 1.60; p = 0.0044, respectively, but not with Type 1 PCV. These findings were further corroborated by another control group in the literature.There may be significantly different associations in genetic variants of elastin between two angiographic phenotypes of PCV.

  14. Synthesis of O-serogroup specific positive controls and real-time PCR standards for nine clinically relevant non-O157 STECs.

    Science.gov (United States)

    Conrad, Cheyenne C; Gilroyed, Brandon H; McAllister, Tim A; Reuter, Tim

    2012-10-01

    Non-O157 Shiga toxin producing Escherichia coli (STEC) are gaining recognition as human pathogens, but no standardized method exists to identify them. Sequence analysis revealed that STEC can be classified on the base of variable O antigen regions into different O serotypes. Polymerase chain reaction is a powerful technique for thorough screening and complex diagnosis for these pathogens, but requires a positive control to verify qualitative and/or quantitative DNA-fragment amplification. Due to the pathogenic nature of STEC, controls are not readily available and cell culturing of STEC reference strains requires biosafety conditions of level 2 or higher. In order to bypass this limitation, controls of stacked O-type specific DNA-fragments coding for primer recognition sites were designed to screen for nine STEC serotypes frequently associated with human infection. The synthetic controls were amplified by PCR, cloned into a plasmid vector and transferred into bacteria host cells. Plasmids amplified by bacterial expression were purified, serially diluted and tested as standards for real-time PCR using SYBR Green and TaqMan assays. Utility of synthetic DNA controls was demonstrated in conventional and real-time PCR assays and validated with DNA from natural STEC strains. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Real-time PCR quantification of six periodontal pathogens in saliva samples from healthy young adults.

    Science.gov (United States)

    Zhou, Xiaodong; Liu, Xiaoli; Li, Jing; Aprecio, Raydolfo M; Zhang, Wu; Li, Yiming

    2015-05-01

    The use of saliva as a diagnostic fluid for the evaluation of periodontal health has gained attention recently. Most published real-time PCR assays focused on quantification of bacteria in subgingival plaque, not in saliva. The aims of this study were to develop a real-time PCR assay for quantification of six periodontal pathogens in saliva and to establish a relationship between the amount of DNA (fg) and colony-forming unit (CFU). TaqMan primers/probe sets were used for the detection of Aggregatibacter actinomycetemcomitans (Aa), Eikenella corrodens (Ec), Fusobacterium nucleatum (Fn), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Tannerella forsythia (Tf), and total bacteria. Six periodontal pathogens and total bacteria in saliva from 24 periodontally healthy individuals were determined. The relationship between the amount of DNA (fg) and CFU was established by measuring the concentrations of extracted bacterial DNA and CFU per milliliter of bacteria on agar plates. Fn, Ec, and Pi were detected in all saliva samples, while 58.5, 45.8, and 33.3% were detected for Tf, Pg, and Aa, respectively. Numbers of Ec and Fn in saliva were highly correlated (R(2) = 0.93, P periodontal pathogens in saliva and estimate the number of live bacteria (CFU). This real-time PCR assay in combination with the relationship between DNA (fg) and CFU has the potential to be an adjunct in evaluation of periodontal health status.

  16. Increased risk for CRC in diabetic patients with the nonrisk allele of SNPs at 8q24.

    Science.gov (United States)

    Ishimaru, Shinya; Mimori, Koshi; Yamamoto, Ken; Inoue, Hiroshi; Imoto, Seiya; Kawano, Shuichi; Yamaguchi, Rui; Sato, Tetsuya; Toh, Hiroyuki; Iinuma, Hisae; Maeda, Toyoki; Ishii, Hideshi; Suzuki, Sadao; Tokudome, Shinkan; Watanabe, Masahiko; Tanaka, Jun-ichi; Kudo, Shin-ei; Sugihara, Ken-ichi; Hase, Kazuo; Mochizuki, Hidetaka; Kusunoki, Masato; Yamada, Kazutaka; Shimada, Yasuhiro; Moriya, Yoshihiro; Barnard, Graham F; Miyano, Satoru; Mori, Masaki

    2012-09-01

    Colorectal cancer (CRC) oncogenesis was considered to be determined by interactions between genetic and environmental factors. Specific interacting factors that influence CRC morbidity have yet to be fully investigated. A multi-institutional collaborative study with 1511 CRC patients and 2098 control subjects was used to compare the odds ratios for the occurrence of polymorphisms at 11 known single nucleotide polymorphisms (SNPs). TaqMan PCR and questionnaires were used to evaluate the effects of environmental exposures. Variants of rs6983267 on 8q24 were the most significant markers of risk for CRC (odds ratio 1.16, 95% confidence interval 1.06-1.27, P = 0.0015). Non-insulin-dependent diabetes mellitus (DM), a higher body mass index at age 20, and meat consumption were environmental risk factors, whereas a tuna-rich diet and vitamin intake were protective factors. The cohort of rs6983267 SNP major (T) allele at 8q24 and DM had a 1.66-fold higher risk ratio than the cohort of major allele patients without DM. We confirmed that interactions between the genetic background and environmental factors are associated with increased risk for CRC. There is a robust risk of the minor G allele at the 8q24 rs6983267 SNP; however, a major T allele SNP could more clearly reveal a correlation with CRC specifically when DM is present.

  17. The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses.

    Science.gov (United States)

    Tydén, E; Löfgren, M; Hakhverdyan, M; Tjälve, H; Larsson, P

    2013-08-01

    In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury. © 2012 John Wiley & Sons Ltd.

  18. An integrated system for identifying the hidden assassins in traditional medicines containing aristolochic acids

    Science.gov (United States)

    Wu, Lan; Sun, Wei; Wang, Bo; Zhao, Haiyu; Li, Yaoli; Cai, Shaoqing; Xiang, Li; Zhu, Yingjie; Yao, Hui; Song, Jingyuan; Cheng, Yung-Chi; Chen, Shilin

    2015-08-01

    Traditional herbal medicines adulterated and contaminated with plant materials from the Aristolochiaceae family, which contain aristolochic acids (AAs), cause aristolochic acid nephropathy. Approximately 256 traditional Chinese patent medicines, containing Aristolochiaceous materials, are still being sold in Chinese markets today. In order to protect consumers from health risks due to AAs, the hidden assassins, efficient methods to differentiate Aristolochiaceous herbs from their putative substitutes need to be established. In this study, 158 Aristolochiaceous samples representing 46 species and four genera as well as 131 non-Aristolochiaceous samples representing 33 species, 20 genera and 12 families were analyzed using DNA barcodes based on the ITS2 and psbA-trnH sequences. Aristolochiaceous materials and their non-Aristolochiaceous substitutes were successfully identified using BLAST1, the nearest distance method and the neighbor-joining (NJ) tree. In addition, based on sequence information of ITS2, we developed a Real-Time PCR assay which successfully identified herbal material from the Aristolochiaceae family. Using Ultra High Performance Liquid Chromatography-Mass Spectrometer (UHPLC-HR-MS), we demonstrated that most representatives from the Aristolochiaceae family contain toxic AAs. Therefore, integrated DNA barcodes, Real-Time PCR assays using TaqMan probes and UHPLC-HR-MS system provides an efficient and reliable authentication system to protect consumers from health risks due to the hidden assassins (AAs).

  19. Comparative miRNA Analysis of Urine Extracellular Vesicles Isolated through Five Different Methods

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    Felix Royo

    2016-12-01

    Full Text Available Urine extracellular vesicles are a valuable low-invasive source of information, especially for the cells of the genitourinary tract. In the search for biomarkers, different techniques have been developed to isolate and characterize the cargo of these vesicles. In the present work, we compare five of these different isolation methods (three commercial isolation kits, ultracentrifugation, and lectin-based purification and perform miRNA profiling using a multiplex miRNA assay. The results showed high correlation through all isolation techniques, and 48 out of 68 miRNAs were detected above the detection limit at least 10 times. The results obtained by multiplex assay were validated through Taqman qPCR. In addition, using this technique combined with a clinically friendly extracellular vesicle (uEV-enrichment method, we performed the analysis of selected miRNAs in urine from patients affected with bladder cancer, benign prostate hyperplasia, or prostate cancer. Importantly, we found that those miRNAs could be detected in almost 100% of the samples, and no significant differences were observed between groups. Our results support the feasibility of analyzing exosomes-associated miRNAs using a methodology that requires a small volume of urine and is compatible with a clinical environment and high-throughput analysis.

  20. Seasonal variation of Legionella in Taiwan's reservoir and its relationships with environmental factors.

    Science.gov (United States)

    Kao, Po-Min; Hsu, Bing-Mu; Chang, Tien-Yu; Hsu, Tsui-Kang; Tzeng, Kai-Jiun; Huang, Yu-Li

    2015-04-01

    In this study, the presence of Legionella in major water reservoirs of Taiwan was examined with respect to seasonal variation, geographical variation, and water quality parameters using TaqMan real-time qPCR. Water samples were collected quarterly at 19 reservoirs in Taiwan between November 2012 and August 2013. The detection rate for Legionella was 35.5% (27/76), and Legionella was detected in all seasons. The Legionella concentration was relatively high in spring and summer, reaching 3.86 × 10(8) and 7.35 × 10(8) cells/L, respectively. By sampling the area, Legionella was detected at a higher proportion in reservoirs in the northern and southern areas, and the difference was consistent in all seasons. Significant association was found between detection of Legionella and various water quality parameters, including conductivity, chlorophyll a, and dissolved oxygen (Mann-Whitney U test, P Legionella detection with pH (P = 0.030, R = -0.497) and dissolved oxygen (P = 0.007, R = -0.596) in fall and positive correlation with Carlson's trophic state index (P = 0.049, R = 0.457) in spring. The identified species included Legionella pneumophila and Legionella drancourtii. The detection of Legionella in reservoirs was indicative of a potential public health risk and should be further evaluated.

  1. MicroRNAs 142-3p, miR-155 and miR-203 Are Deregulated in Gastric MALT Lymphomas Compared to Chronic Gastritis.

    Science.gov (United States)

    Fernández, Concepción; Bellosillo, Beatriz; Ferraro, Mariana; Seoane, Agustín; Sánchez-González, Blanca; Pairet, Silvia; Pons, Aina; Barranco, Luis; Vela, María Carmen; Gimeno, Eva; Colomo, Lluís; Besses, Carles; Navarro, Alfons; Salar, Antonio

    2017-01-02

    Over the last years, our knowledge on pathogenesis of gastric MALT lymphoma has greatly improved, but its morphological diagnosis is still hampered by overlapping histological features with advanced chronic gastritis. MicroRNAs are deregulated in lymphomas, but their role and usefulness in gastric MALT lymphoma has not been extensively investigated. We analyzed the expression of 384 miRNAs using TaqMan microRNA assay in a training series of 10 gastric MALT lymphomas, 3 chronic gastritis and 2 reactive lymph nodes. Then, significantly deregulated miRNAs were individually assessed by real-time PCR in a validation series of 16 gastric MALT lymphomas and 12 chronic gastritis. Gastric MALT lymphoma is characterized by a specific miRNA expression profile. Among the differentially expressed miRNAs, a significant overexpression of miR-142-3p and miR-155 and down-regulation of miR-203 was observed in gastric MALT lymphoma when compared to chronic gastritis. miR-142-3p, miR-155 and miR-203 expression levels might be helpful biomarkers for the differential diagnosis between gastric MALT lymphomas and chronic gastritis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  2. PCR-based detection of gene transfer vectors: application to gene doping surveillance.

    Science.gov (United States)

    Perez, Irene C; Le Guiner, Caroline; Ni, Weiyi; Lyles, Jennifer; Moullier, Philippe; Snyder, Richard O

    2013-12-01

    Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.

  3. Detection of growth hormone doping by gene expression profiling of peripheral blood.

    Science.gov (United States)

    Mitchell, Christopher J; Nelson, Anne E; Cowley, Mark J; Kaplan, Warren; Stone, Glenn; Sutton, Selina K; Lau, Amie; Lee, Carol M Y; Ho, Ken K Y

    2009-12-01

    GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration. Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans. Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes. GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men. Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

  4. Let7a involves in neural stem cell differentiation relating with TLX level

    Energy Technology Data Exchange (ETDEWEB)

    Song, Juhyun [Department of Anatomy, Yonsei University College of Medicine, Seoul (Korea, Republic of); Cho, Kyoung Joo; Oh, Yumi [Department of Anatomy, Yonsei University College of Medicine, Seoul (Korea, Republic of); BK21 Plus Project for Medical Sciences, and Brain Research Institute, Yonsei University College of Medicine, Seoul (Korea, Republic of); Lee, Jong Eun, E-mail: jelee@yuhs.ac [Department of Anatomy, Yonsei University College of Medicine, Seoul (Korea, Republic of); BK21 Plus Project for Medical Sciences, and Brain Research Institute, Yonsei University College of Medicine, Seoul (Korea, Republic of)

    2015-07-10

    Neural stem cells (NSCs) have the potential for differentiation into neurons known as a groundbreaking therapeutic solution for central nervous system (CNS) diseases. To resolve the therapeutic efficiency of NSCs, recent researchers have focused on the study on microRNA's role in CNS. Some micro RNAs have been reported significant functions in NSC self-renewal and differentiation through the post-transcriptional regulation of neurogenesis genes. MicroRNA-Let7a (Let7a) has known as the regulator of diverse cellular mechanisms including cell differentiation and proliferation. In present study, we investigated whether Let7a regulates NSC differentiation by targeting the nuclear receptor TLX, which is an essential regulator of NSC self-renewal, proliferation and differentiation. We performed the following experiments: western blot analysis, TaqMan assay, RT-PCR, and immunocytochemistry to confirm the alteration of NSCs. Our data showed that let7a play important roles in controlling NSC fate determination. Thus, manipulating Let-7A and TLX could be a novel strategy to enhance the efficiency of NSC's neuronal differentiation for CNS disorders. - Highlights: • Let7a influences on NSC differentiation and proliferation. • Let7a involves in mainly NSC differentiation rather than proliferation. • Let7a positively regulates the TLX expression.

  5. Nonassociation of homocysteine gene polymorphisms with treatment outcome in South Indian Tamil Rheumatoid Arthritis patients.

    Science.gov (United States)

    Muralidharan, Niveditha; Gulati, Reena; Misra, Durga Prasanna; Negi, Vir S

    2018-02-01

    The aim of the study was to look for any association of MTR 2756A>G and MTRR 66A>G gene polymorphisms with clinical phenotype, methotrexate (MTX) treatment response, and MTX-induced adverse events in South Indian Tamil patients with rheumatoid arthritis (RA). A total of 335 patients with RA were investigated. MTR 2756A>G gene polymorphism was analyzed by PCR-RFLP, and MTRR 66A>G SNP was analyzed by TaqMan 5' nuclease assay. The allele frequencies were compared with HapMap groups. MTR 2756G allele was found to be associated with risk of developing RA. The allele frequencies of MTR 2756A>G and MTRR 66A>G SNPs in controls differed significantly when compared with HapMap groups. Neither of the SNPs influenced the MTX treatment outcome and adverse effects. Neither of the SNPs seems to be associated with MTX treatment outcome and adverse events in South Indian Tamil patients with RA.

  6. Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

    Science.gov (United States)

    Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

    2003-12-15

    Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

  7. Real-time PCR genotyping assay for canine progressive rod-cone degeneration and mutant allele frequency in Toy Poodles, Chihuahuas and Miniature Dachshunds in Japan.

    Science.gov (United States)

    Kohyama, Moeko; Tada, Naomi; Mitsui, Hiroko; Tomioka, Hitomi; Tsutsui, Toshihiko; Yabuki, Akira; Rahman, Mohammad Mahbubur; Kushida, Kazuya; Mizukami, Keijiro; Yamato, Osamu

    2016-03-01

    Canine progressive rod-cone degeneration (PRCD) is a middle- to late-onset, autosomal recessive, inherited retinal disorder caused by a substitution (c.5G>A) in the canine PRCD gene that has been identified in 29 or more purebred dogs. In the present study, a TaqMan probe-based real-time PCR assay was developed and evaluated for rapid genotyping and large-scale screening of the mutation. Furthermore, a genotyping survey was carried out in a population of the three most popular breeds in Japan (Toy Poodles, Chihuahuas and Miniature Dachshunds) to determine the current mutant allele frequency. The assay separated all the genotypes of canine PRCD rapidly, indicating its suitability for large-scale surveys. The results of the survey showed that the mutant allele frequency in Toy Poodles was high enough (approximately 0.09) to allow the establishment of measures for the prevention and control of this disorder in breeding kennels. The mutant allele was detected in Chihuahuas for the first time, but the frequency was lower (approximately 0.02) than that in Toy Poodles. The mutant allele was not detected in Miniature Dachshunds. This assay will allow the selective breeding of dogs from the two most popular breeds (Toy Poodle and Chihuahua) in Japan and effective prevention or control of the disorder.

  8. Association of the C-Reactive Protein Gene (CRP rs1205 C>T Polymorphism with Aortic Valve Calcification in Patients with Aortic Stenosis

    Directory of Open Access Journals (Sweden)

    Ewa Wypasek

    2015-10-01

    Full Text Available Elevation in C-reactive protein (CRP levels have been shown in patients with aortic valve stenosis (AS. Minor allele of the CRP gene (CRP rs1205 C>T polymorphism has been associated with lower plasma CRP concentrations in cohorts of healthy and atherosclerotic patients. Considering the existing similarities between atherosclerosis and AS, we examined the effect of CRP rs1205 C>T polymorphism on the AS severity. Three hundred consecutive Caucasian patients diagnosed with AS were genotyped for the rs1205 C>T polymorphism using the TaqMan assay. Severity of the AS was assessed using transthoracic echocardiography. The degree of calcification was analyzed semi-quantitatively. Carriers of the rs1205 T allele were characterized by elevated serum CRP levels (2.53 (1.51–3.96 vs. 1.68 (0.98–2.90 mg/L, p < 0.001 and a higher proportion of the severe aortic valve calcification (70.4% vs. 55.1%, p = 0.01 compared with major homozygotes. The effect of CRP rs1205 polymorphism on CRP levels is opposite in AS-affected than in unaffected subjects, suggesting existence of a disease-specific molecular regulatory mechanism. Furthermore, rs1205 variant allele predisposes to larger aortic valve calcification, potentially being a novel genetic risk marker of disease progression.

  9. Novel Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Varroa destructor Populations from the Southeastern USA.

    Directory of Open Access Journals (Sweden)

    Joel González-Cabrera

    Full Text Available The parasitic mite Varroa destructor has a significant worldwide impact on bee colony health. In the absence of control measures, parasitized colonies invariably collapse within 3 years. The synthetic pyrethroids tau-fluvalinate and flumethrin have proven very effective at managing this mite within apiaries, but intensive control programs based mainly on one active ingredient have led to many reports of pyrethroid resistance. In Europe, a modification of leucine to valine at position 925 (L925V of the V. destructor voltage-gated sodium channel was correlated with resistance, the mutation being found at high frequency exclusively in hives with a recent history of pyrethroid treatment. Here, we identify two novel mutations, L925M and L925I, in tau-fluvalinate resistant V. destructor collected at seven sites across Florida and Georgia in the Southeastern region of the USA. Using a multiplexed TaqMan® allelic discrimination assay, these mutations were found to be present in 98% of the mites surviving tau-fluvalinate treatment. The mutations were also found in 45% of the non-treated mites, suggesting a high potential for resistance evolution if selection pressure is applied. The results from a more extensive monitoring programme, using the Taqman® assay described here, would clearly help beekeepers with their decision making as to when to include or exclude pyrethroid control products and thereby facilitate more effective mite management programmes.

  10. Lack of association between KCNJ11 (rs5219 and ABCC8 (rs757110 polymorphisms and sulphonylurea treatment response in type 2 diabetes patients in Novosibirsk region

    Directory of Open Access Journals (Sweden)

    Irina Arkad'evna Bondar

    2015-03-01

    Full Text Available Aim. Sulfonylureas (SU are widely used in everyday clinical practice in treatment of patients with type 2 diabetes mellitus (T2DM. There is a considerable variability in SU effects, which may be caused by psychological, social, biological and genetic factors. The aim of the study was to investigate the association between rs5219 KCNJ11 gene and rs757110 ABCC8 gene polymorphism and long-term response to SU-drugs of second and third generation in the Novosibirsk region. Materials and Methods. 326 patients with type 2 diabetes in the Novosibirsk region were examined. Patients were divided into 2 groups, depending on HbA1c level. The first group included patients with target HbA1c levels on SU monotherapy. The second group included patients who did not reach target HbA1c levels on the highest dose of SU. Genotyping of KCNJ11 (rs5219 and ABCC8 (rs757110 was performed by TaqMan real-time PCR (ICBFM SB RAS, Novosibirsk, Russia. Results. Patients with type 2 diabetes with a good response to SU-therapy compared to the group of patients with a poor response to SU-therapy were older (65.8?9.1 years vs. 61.6?7.9 years, p

  11. Parallel evolution under chemotherapy pressure in 29 breast cancer cell lines results in dissimilar mechanisms of resistance.

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    Bálint Tegze

    Full Text Available BACKGROUND: Developing chemotherapy resistant cell lines can help to identify markers of resistance. Instead of using a panel of highly heterogeneous cell lines, we assumed that truly robust and convergent pattern of resistance can be identified in multiple parallel engineered derivatives of only a few parental cell lines. METHODS: Parallel cell populations were initiated for two breast cancer cell lines (MDA-MB-231 and MCF-7 and these were treated independently for 18 months with doxorubicin or paclitaxel. IC50 values against 4 chemotherapy agents were determined to measure cross-resistance. Chromosomal instability and karyotypic changes were determined by cytogenetics. TaqMan RT-PCR measurements were performed for resistance-candidate genes. Pgp activity was measured by FACS. RESULTS: All together 16 doxorubicin- and 13 paclitaxel-treated cell lines were developed showing 2-46 fold and 3-28 fold increase in resistance, respectively. The RT-PCR and FACS analyses confirmed changes in tubulin isofom composition, TOP2A and MVP expression and activity of transport pumps (ABCB1, ABCG2. Cytogenetics showed less chromosomes but more structural aberrations in the resistant cells. CONCLUSION: We surpassed previous studies by parallel developing a massive number of cell lines to investigate chemoresistance. While the heterogeneity caused evolution of multiple resistant clones with different resistance characteristics, the activation of only a few mechanisms were sufficient in one cell line to achieve resistance.

  12. Development of FQ-PCR method to determine the level of ADD1 expression in fatty and lean pigs.

    Science.gov (United States)

    Cui, J X; Chen, W; Zeng, Y Q

    2015-10-30

    To determine how adipocyte determination and differentiation factor 1 (ADD1), a gene involved in the determination of pork quality, is regulated in Laiwu and Large pigs, we used TaqMan fluorescence quantitative real-time polymerase chain reaction (FQ-PCR) to detect differential expression in the longissimus muscle of Laiwu (fatty) and Large White (lean) pigs. In this study, the ADD1 and GAPDH cDNA sequences were cloned using a T-A cloning assay, and the clone sequences were consistent with those deposited in GenBank. Thus, the target fragment was successfully recombined into the vector, and its integrity was maintained. The standard curve and regression equation were established through the optimized FQ-PCR protocol. The standard curve of porcine ADD1 and GAPDH cDNA was determined, and its linear range extension could reach seven orders of magnitudes. The results showed that this method was used to quantify ADD1 expression in the longissimus muscle of two breeds of pig, and was found to be accurate, sensitive, and convenient. These results provide information regarding porcine ADD1 mRNA expression and the mechanism of adipocyte differentiation, and this study could help in the effort to meet the demands of consumers interested in the maintenance of health and prevention of obesity. Furthermore, it could lead to new approaches in the prevention and clinical treatment of this disease.

  13. Cytokine mRNA profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: Association of sustained expression of IFN-gamma and IL-10 after viral clearance

    DEFF Research Database (Denmark)

    Johnsen, C. K.; Bøtner, Anette; Kamstrup, Søren

    2002-01-01

    An experimental model was used to investigate mRNA cytokine profiles in bronchoalvolar cells (BALC) from piglets, infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). The BALC's were analyzed for the cytokines TNF-alpha, IFN-gamma, IL-8, IL-10, and IL-12(p40) by real......-time TaqMan polymerase chain reaction in 2-, 4-, and 6-week-old piglets, respectively. High levels of IFN-gamma mRNA was detected in all piglets, while IL-10 was upregulated in 2-week-old piglets, was at normal levels in 4-week-old piglets, and elevated again in 6-week-old piglets. IL-12 was weakly...... elevated in all three age groups. Virus was reduced by 50% in 4-week-old piglets and cleared by 6 weeks of age. The sustained expression of IFNgamma and reduction of IL-10 production indicate an important role for these cytokines in immunity to PRRSV....

  14. Validation of a Real Time PCR for Classical Swine Fever Diagnosis

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    Natanael Lamas Dias

    2014-01-01

    Full Text Available The viral disease classical swine fever (CSF, caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5′NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5′NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.

  15. Validation of a real time PCR for classical Swine Fever diagnosis.

    Science.gov (United States)

    Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Erica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves; Camargos, Marcelo Fernandes

    2014-01-01

    The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5'NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5'NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.

  16. Differential expression of miRNAs and their relation to active tuberculosis.

    Science.gov (United States)

    Xu, Zhihong; Zhou, Aiping; Ni, Jinjing; Zhang, Qiufen; Wang, Ying; Lu, Jie; Wu, Wenjuan; Karakousis, Petros C; Lu, Shuihua; Yao, Yufeng

    2015-07-01

    The aim of this work was to screen miRNA signatures dysregulated in tuberculosis to improve our understanding of the biological role of miRNAs involved in the disease. Datasets deposited in publically available databases from microarray studies on infectious diseases and malignancies were retrieved, screened, and subjected to further analysis. Effect sizes were combined using the inverse-variance model and between-study heterogeneity was evaluated by the random effects model. 35 miRNAs were differentially expressed (12 up-regulated, 23 down-regulated; p tuberculosis and other infectious diseases. 15 miRNAs were found to be significantly differentially regulated (7 up-regulated, 8 down-regulated; p tuberculosis and malignancies. Most of the miRNA signatures identified in this study were found to be involved in immune responses and metabolism. Expression of these miRNA signatures in serum samples from TB subjects (n = 11) as well as healthy controls (n = 10) was examined by TaqMan miRNA array. Taken together, the results revealed differential expression of miRNAs in TB, but available datasets are limited and these miRNA signatures should be validated in future studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Improved quantification accuracy for duplex real-time PCR detection of genetically modified soybean and maize in heat processed foods

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    CHENG Fang

    2013-04-01

    Full Text Available Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several real-time PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR was further validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.

  18. Use of allele-specific FAIRE to determine functional regulatory polymorphism using large-scale genotyping arrays.

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    Andrew J P Smith

    Full Text Available Following the widespread use of genome-wide association studies (GWAS, focus is turning towards identification of causal variants rather than simply genetic markers of diseases and traits. As a step towards a high-throughput method to identify genome-wide, non-coding, functional regulatory variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α, rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006, and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly comprehensive genotyping chips and distinct tissues for examination, FAIRE-gen has the potential to aid the identification of many causal SNPs associated with disease from GWAS.

  19. Significance of common variants on human chromosome 8q24 in relation to the risk of prostate cancer in native Japanese men

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    Hosoi Takayuki

    2009-07-01

    Full Text Available Abstract Background Common variants on human chromosome 8q24, rs1447295 (C/A and rs6983267 (T/G, have been recently linked to the prevalence of prostate cancer in European and American populations. Here, we evaluated whether the single-nucleotide polymorphisms rs1447295 and rs6983267 were associated with the risk of sporadic prostate cancer as well as latent prostate cancer in a native Japanese population. Results We analyzed genomic DNA samples from 391 sporadic prostate cancer patients, 323 controls who had died from causes unrelated to cancer and 112 Japanese men who were diagnosed as having latent prostate cancer based on autopsy results. The polymorphisms were determined by allelic discrimination using a fluorescent-based TaqMan assay. The A allele of rs1447295 was significantly associated with the risk of sporadic prostate cancer (p = 0.04; age-adjusted OR, 1.34, while the G allele of rs6983267 showed a trend towards being a high-risk allele (p = 0.06; age-adjusted OR, 1.27. No significant difference between these two polymorphisms and the risk of latent prostate cancer was observed in the present Japanese population. Conclusion Known variants on human chromosome 8q24 may be risk factors for sporadic prostate cancer in native Japanese men.

  20. The Association between Single-Nucleotide Polymorphisms of ORAI1 Gene and Breast Cancer in a Taiwanese Population

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    Wei-Chiao Chang

    2012-01-01

    Full Text Available Background. Breast cancer is the most common malignancy in women. There is increasing evidence suggesting that ORAI1, components of store-operated calcium channel, play a pivotal role in breast cancer progression and metastasis. Methods. A total of 384 female patients with breast cancer were included in this study. We selected five representative tagging ORAI1 SNPs from HapMap database with minimum allele frequency (MAF >10%. Genotyping was performed using TaqMan allelic discrimination assay. Chi-square (χ2 test was used to analyze statistical differences among control and patient groups in genotype and allelic frequencies. Results. Two of the ORAI1 SNPs (rs12320939 and rs12313273 were associated with estrogen receptors positive in breast cancer patients under the recessive model. When the Bonferroni correction was performed, the significance still existed. In addition, rs12320939 also associated with the lymph nodal involvement. Conclusion. We showed that genetic polymorphisms of ORAI1 associated strongly with lymph nodal involvement and estrogen receptors (ERs positive breast cancer patients in a Taiwanese population.

  1. A Single Nucleotide Polymorphism (rs4236480 in TRPV5 Calcium Channel Gene Is Associated with Stone Multiplicity in Calcium Nephrolithiasis Patients

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    Anas Khaleel

    2015-01-01

    Full Text Available Nephrolithiasis is characterized by calcification of stones in the kidneys from an unknown cause. Animal models demonstrated the functional roles of the transient receptor potential vanilloid member 5 (TRPV5 gene in calcium renal reabsorption and hypercalciuria. Therefore, TRPV5 was suggested to be involved in calcium homeostasis. However, whether genetic polymorphisms of TRPV5 are associated with kidney stone multiplicity or recurrence is unclear. In this study, 365 Taiwanese kidney-stone patients were recruited. Both biochemical data and DNA samples were collected. Genotyping was performed by a TaqMan allelic discrimination assay. We found that a TRPV5 polymorphism (rs4236480 was observed to be associated with stone multiplicity of calcium nephrolithiasis, as the risk of stone multiplicity was higher in patients with the TT+CT genotype than in patients with the CC genotype (p=0.0271. In summary, despite the complexity of nephrolithiasis and the potential association of numerous calcium homeostatic absorption/reabsorption factors, TRPV5 plays an important role in the pathogenesis of calcium nephrolithiasis.

  2. Association of caspase-1 polymorphisms with Chagas cardiomyopathy among individuals in Santa Cruz, Bolivia.

    Science.gov (United States)

    Fu, Katherine Yih-Jia; Zamudio, Roxana; Henderson-Frost, Jo; Almuedo, Alex; Steinberg, Hannah; Clipman, Steven Joseph; Duran, Gustavo; Marcus, Rachel; Crawford, Thomas; Alyesh, Daniel; Colanzi, Rony; Flores, Jorge; Gilman, Robert Hugh; Bern, Caryn

    2017-01-01

    Trypanosoma cruzi (Tc) infection is usually acquired in childhood in endemic areas, leading to Chagas disease, which progresses to Chagas cardiomyopathy in 20-30% of infected individuals over decades. The pathogenesis of Chagas cardiomyopathy involves the host inflammatory response to T. cruzi, in which upstream caspase-1 activation prompts the cascade of inflammatory chemokines/cytokines, cardiac remodeling, and myocardial dysfunction. The aim of the present study was to examine the association of two caspase-1 single nucleotide polymorphisms (SNPs) with cardiomyopathy. We recruited infected (Tc+, n = 149) and uninfected (Tc-, n = 87) participants in a hospital in Santa Cruz, Bolivia. Cardiac status was classified (I, II, III, IV) based on Chagas cardiomyopathy-associated electrocardiogram findings and ejection fractions on echocardiogram. Genotypes were determined using Taqman probes via reverse transcription-polymerase chain reaction of peripheral blood DNA. Genotype frequencies were analyzed according to three inheritance patterns (dominant, recessive, additive) using logistic regression adjusted for age and sex. The AA allele for the caspase-1 SNP rs501192 was more frequent in Tc+ cardiomyopathy (classes II, III, IV) patients compared to those with a normal cardiac status (class I) [odds ratio (OR) = -2.18, p = 0.117]. This trend approached statistical significant considering only Tc+ patients in class I and II (OR = -2.64, p = 0.064). Caspase-1 polymorphisms may play a role in Chagas cardiomyopathy development and could serve as markers to identify individuals at higher risk for priority treatment.

  3. Qat Chewing and Periodontal Pathogens in Health and Disease: Further Evidence for a Prebiotic-Like Effect

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    Abdulrahman Al-Alimi

    2015-01-01

    Full Text Available Aim. Qat chewing has been reported to induce subgingival microbial shifts suggestive of prebiotic-like properties. The objective here was to assess the effect of qat chewing on a panel of classical and new putative periopathogens in health and periodontitis. Materials and Methods. 40 qat chewers and 40 nonchewers, equally stratified by periodontal health status, were recruited. Taqman, real-time PCR was used to quantify total bacteria, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Parvimonas micra, Filifactor alocis, Synergistetes, and TM7s in pooled subgingival biofilm samples. Differences in microbial parameters between the study groups were analysed using ordinal regression. Results. In health, the qat chewers harboured significantly lower relative counts of P. gingivalis, T. forsythia, Synergistetes, and TM7s after adjustment for multiple comparisons (P≤0.007. At nominal significance level, they also carried lower counts of TM7s and P. micra (P≤0.05. In periodontitis, the chewers had lower counts of all taxa; however, only T. denticola withstood correction for multiple comparisons (P≤0.0063. Conclusions. Qat chewing is associated with lower proportions of periopathogens, particularly in subjects with healthy periodontium, which supports previous reports of its prebiotic-like properties. This potentially beneficial biological effect can be exploited by attempting to isolate the active fraction.

  4. HCV infection among Saudi population: high prevalence of genotype 4 and increased viral clearance rate.

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    Ahmed S Abdel-Moneim

    Full Text Available HCV is a major etiological agent of liver disease with a high rate of chronic evolution. The virus possesses 6 genotypes with many subtypes. The rate of spontaneous clearance among HCV infected individuals denotes a genetic determinant factor. The current study was designed in order to estimate the rate of HCV infection and ratio of virus clearance among a group of infected patients in Saudi Arabia from 2008 to 2011. It was additionally designed to determine the genotypes of the HCV in persistently infected patients. HCV seroprevalence was conducted on a total of 15,323 individuals. Seropositive individuals were tested by Cobas AmpliPrep/Cobas TaqMan HCV assay to determine the ratio of persistently infected patients to those who showed spontaneous viral clearance. HCV genotyping on random samples from persistently infected patients were conducted based on the differences in the 5'untranslated region (5'UTR. Anti-HCV antibodies were detected in 7.3% of the totally examined sera. A high percentage of the HCV infected individuals experienced virus clearance (48.4%. HCV genotyping revealed the presence of genotypes 1 and 4, the latter represented 97.6% of the tested strains. Evidences of the widespread of the HCV genotype 4 and a high rate of HCV virus clearance were found in Saudi Arabia.

  5. Comprehensive analysis to explain reduced or increased SOD1 enzymatic activity in ALS patients and their relatives.

    Science.gov (United States)

    Keskin, Isil; Birve, Anna; Berdynski, Mariusz; Hjertkvist, Karin; Rofougaran, Reza; Nilsson, Torbjörn K; Glass, Jonathan D; Marklund, Stefan L; Andersen, Peter M

    2017-08-01

    To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72 HRE . In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.

  6. The association of age-related maculopathy susceptibility 2 (ARMS2) and complement factor H (CFH) variants with two angiographic subtypes of polypoidal choroidal vasculopathy.

    Science.gov (United States)

    Miki, Akiko; Honda, Shigeru; Kondo, Naoshi; Negi, Akira

    2013-09-01

    To compare the association of age-related maculopathy susceptibility 2 (ARMS2) and complement factor H (CFH) variants between two different angiographic phenotypes of polypoidal choroidal vasculopathy (PCV). We included 175 Japanese patients with PCV and 150 age- and sex-matched controls. PCV was classified into two phenotypes (Type 1 and Type 2) according to the presence or absence of the feeding vessels found in indocyanine-green angiography. The single nucleotide polymorphism (SNP) at rs10490924 (A69S) in ARMS2 and rs800292 (I62V), rs1061170 (Y402H) in the CFH region were genotyped using the TaqMan assay. The minor allele frequency (MAF) of rs10490924 was significantly different between Type 1 PCV (n = 81) and control (p < 0.0001), while no difference was found between Type 2 PCV (n = 94) and control (p = 0.20). The MAF of rs800292 was significantly different between each type of PCV and control (p < 0.0001 and 0.0001 for Type 1 versus control and Type 2 versus control, respectively). The MAF of rs1061170 was not significantly different between either type of PCV and control (p = 0.084 and 0.15, respectively). There may be significantly different associations in the genetic variants of ARMS2 between two angiographic phenotypes of PCV.

  7. A real-time PCR assay for the relative quantification of the tetrahydrocannabinolic acid (THCA) synthase gene in herbal Cannabis samples.

    Science.gov (United States)

    Cascini, Fidelia; Passerotti, Stella; Martello, Simona

    2012-04-10

    In this study, we wanted to investigate whether or not the tetrahydrocannabinolic acid (THCA) synthase gene, which codes for the enzyme involved in the biosynthesis of THCA, influences the production and storage of tetrahydrocannabinol (THC) in a dose-dependent manner. THCA is actually decarboxylated to produce THC, the main psychoactive component in the Cannabis plant. Assuming as the research hypothesis a correlation between the gene copy number and the production of THC, gene quantification could be useful in forensics in order to complement or replace chemical analysis for the identification and classification of seized Cannabis samples, thus distinguishing the drug-type from the fibre-type varieties. A real-time PCR assay for the relative quantification of the THCA synthase gene was then validated on Cannabis samples; some were seized from the illegal drug market and others were derived from experimental cultivation. In order to determine the gene copy number to compare high vs. low potency plants, we chose the ΔΔCt method for TaqMan reactions. The assay enabled single plants with zero, one, and two copies of the gene to be distinguished. As a result of this first part of the research on the THCA synthase gene (the second part will cover a study of gene expression), we found no correlation between THCA synthase gene copy number and the content of THC in the herbal Cannabis samples tested. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  8. The progesterone receptor Val660→Leu polymorphism and breast cancer risk

    International Nuclear Information System (INIS)

    De Vivo, Immaculata; Hankinson, Susan E; Colditz, Graham A; Hunter, David J

    2004-01-01

    Recent evidence suggests a role for progesterone in breast cancer development and tumorigenesis. Progesterone exerts its effect on target cells by interacting with its receptor; thus, genetic variations, which might cause alterations in the biological function in the progesterone receptor (PGR), can potentially contribute to an individual's susceptibility to breast cancer. It has been reported that the PROGINS allele, which is in complete linkage disequilibrium with a missense substitution in exon 4 (G/T, valine→leucine, at codon 660), is associated with a decreased risk for breast cancer. Using a nested case-control study design within the Nurses' Health Study cohort, we genotyped 1252 cases and 1660 matched controls with the use of the Taqman assay. We did not observe any association of breast cancer risk with carrying the G/T (Val660→Leu) polymorphism (odds ratio 1.10, 95% confidence interval 0.93–1.30). In addition, we did not observe an interaction between this allele and menopausal status and family history of breast cancer as reported previously. Overall, our study does not support an association between the Val660→Leu PROGINS polymorphism and breast cancer risk

  9. Role of canine circovirus in dogs with acute haemorrhagic diarrhoea.

    Science.gov (United States)

    Anderson, A; Hartmann, K; Leutenegger, C M; Proksch, A L; Mueller, R S; Unterer, S

    2017-06-03

    Canine circovirus (CanineCV) has been detected in some dogs with severe haemorrhagic diarrhoea, but its pathogenic role is unclear. This study evaluated a suspected association between the presence of CanineCV and acute haemorrhagic diarrhoea syndrome (AHDS) in dogs. The prevalence of CanineCV in dogs with AHDS was compared with that in healthy dogs and those infected with canine parvovirus (CPV). Additionally, time to recovery and mortality rate were compared between CanineCV-positive and CanineCV-negative dogs. Faecal samples of dogs with AHDS (n=55), healthy dogs (n=66) and dogs infected with CPV (n=54) were examined by two real-time TaqMan PCR assays targeting the replicase and capsid genes of CanineCV. CanineCV was detected in faecal samples of two dogs with AHDS, three healthy controls and seven dogs infected with CPV. Among the three groups, there was no significant difference in prevalence of CanineCV. CPV-infected animals that were coinfected with CanineCV had a significantly higher mortality rate compared with those negative for CanineCV. CanineCV does not appear to be the primary causative agent of AHDS in dogs, but might play a role as a negative co-factor in disease outcome in dogs with CPV infection. British Veterinary Association.

  10. Impacts of CA9 gene polymorphisms on urothelial cell carcinoma susceptibility and clinicopathologic characteristics in Taiwan.

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    Shian-Shiang Wang

    Full Text Available Carbonic anhydrase 9 (CA9 is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study explored the effect of CA9 gene polymorphisms on the susceptibility of developing urothelial cell carcinoma (UCC and the clinicopathological status.A total of 442 participants, including 221 healthy people and 221 patients with UCC, were recruited for this study. Four single-nucleotide polymorphisms (SNPs of the CA9 gene were assessed by a real-time PCR with the TaqMan assay. After adjusting for other co-variants, the individuals carrying at least one A allele at CA9 rs1048638 had a 2.303-fold risk of developing UCC than did wild-type (CC carriers. Furthermore, UCC patients who carried at least one A allele at rs1048638 had a higher invasive stage risk (p< 0.05 than did patients carrying the wild-type allele. Moreover, among the UCC patients with smoker, people with at least one A allele of CA9 polymorphisms (rs1048638 had a 4.75-fold (95% CI = 1.204-18.746 increased risk of invasive cancer.The rs1048638 polymorphic genotypes of CA9 might contribute to the prediction of susceptibility to and pathological development of UCC. This is the first study to provide insight into risk factors associated with CA9 variants in carcinogenesis of UCC in Taiwan.

  11. Over-Expression of Dopamine D2 Receptor and Inwardly Rectifying Potassium Channel Genes in Drug-Naive Schizophrenic Peripheral Blood Lymphocytes as Potential Diagnostic Markers

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    Ágnes Zvara

    2005-01-01

    Full Text Available Schizophrenia is one of the most common neuropsychiatric disorders affecting nearly 1% of the human population. Current diagnosis of schizophrenia is based on complex clinical symptoms. The use of easily detectable peripheral molecular markers could substantially help the diagnosis of psychiatric disorders. Recent studies showed that peripheral blood lymphocytes (PBL express subtypes of D1 and D2 subclasses of dopamine receptors. Recently, dopamine receptor D3 (DRD3 was found to be over-expressed in schizophrenic PBL and proposed to be a diagnostic and follow-up marker for schizophrenia. In this study we screened PBL of 13 drug-naive/drug-free schizophrenic patients to identify additional markers of schizophrenia. One of the benefits of our study is the use of blood samples of non-medicated, drug-naive patients. This excludes the possibility that changes detected in gene expression levels might be attributed to the medication rather than to the disorder itself. Among others, genes for dopamine receptor D2 (DRD2 and the inwardly rectifying potassium channel (Kir2.3 were found to be over-expressed in microarray analysis. Increased mRNA levels were confirmed by quantitative real-time PCR (QRT-PCR using the SybrGreen method and dual labeled TaqMan probes. The use of both molecular markers allows a more rapid and precise prediction of schizophrenia and might help find the optimal medication for schizophrenic patients.

  12. Market analysis of food products for detection of allergenic walnut (Juglans regia) and pecan (Carya illinoinensis) by real-time PCR.

    Science.gov (United States)

    López-Calleja, Inés María; de la Cruz, Silvia; González, Isabel; García, Teresa; Martín, Rosario

    2015-06-15

    Two real-time polymerase chain reaction (PCR)-based assays for detection of walnut (Juglans regia) and pecan (Carya illinoinensis) traces in a wide range of processed foods are described here. The method consists on a real-time PCR assay targeting the ITS1 region, using a nuclease (TaqMan) probe labeled with FAM and BBQ. The method was positive for walnut and pecan respectively, and negative for all other heterologous plants and animals tested. Using a series of model samples with defined raw walnut in wheat flour and heat-treated walnut in wheat flour with a range of concentrations of 0.1-100,000 mg kg(-1), a practical detection limit of 0.1 mg kg(-1) of walnut content was estimated. Identical binary mixtures were done for pecan, reaching the same limit of detection of 0.1 mg kg(-1). The assay was successfully trialed on a total of 232 commercial foodstuffs. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. eCOMPAGT – efficient Combination and Management of Phenotypes and Genotypes for Genetic Epidemiology

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    Specht Günther

    2009-05-01

    Full Text Available Abstract Background High-throughput genotyping and phenotyping projects of large epidemiological study populations require sophisticated laboratory information management systems. Most epidemiological studies include subject-related personal information, which needs to be handled with care by following data privacy protection guidelines. In addition, genotyping core facilities handling cooperative projects require a straightforward solution to monitor the status and financial resources of the different projects. Description We developed a database system for an efficient combination and management of phenotypes and genotypes (eCOMPAGT deriving from genetic epidemiological studies. eCOMPAGT securely stores and manages genotype and phenotype data and enables different user modes with different rights. Special attention was drawn on the import of data deriving from TaqMan and SNPlex genotyping assays. However, the database solution is adjustable to other genotyping systems by programming additional interfaces. Further important features are the scalability of the database and an export interface to statistical software. Conclusion eCOMPAGT can store, administer and connect phenotype data with all kinds of genotype data and is available as a downloadable version at http://dbis-informatik.uibk.ac.at/ecompagt.

  14. Establishment of a universal and rational gene detection strategy through three-way junction-based remote transduction.

    Science.gov (United States)

    Tang, Yidan; Lu, Baiyang; Zhu, Zhentong; Li, Bingling

    2018-01-21

    The polymerase chain reaction and many isothermal amplifications are able to achieve super gene amplification. Unfortunately, most commonly-used transduction methods, such as dye staining and Taqman-like probing, still suffer from shortcomings including false signals or difficult probe design, or are incompatible with multi-analysis. Here a universal and rational gene detection strategy has been established by translating isothermal amplicons to enzyme-free strand displacement circuits via three-way junction-based remote transduction. An assistant transduction probe was imported to form a partial hybrid with the target single-stranded nucleic acid. After systematic optimization the hybrid could serve as an associative trigger to activate a downstream circuit detector via a strand displacement reaction across the three-way junction. By doing so, the detection selectivity can be double-guaranteed through both amplicon-transducer recognition and the amplicon-circuit reaction. A well-optimized circuit can be immediately applied to a new target detection through simply displacing only 10-12 nt on only one component, according to the target. More importantly, this property for the first time enables multi-analysis and logic-analysis in a single reaction, sharing a single fluorescence reporter. In an applicable model, trace amounts of Cronobacter and Enterobacteria genes have been clearly distinguished from samples with no bacteria or one bacterium, with ultra-high sensitivity and selectivity.

  15. Valacyclovir pharmacokinetics and exploratory pharmacodynamics in young adults with Epstein-Barr virus infectious mononucleosis.

    Science.gov (United States)

    Vezina, Heather E; Balfour, Henry H; Weller, Dennis R; Anderson, Bruce J; Brundage, Richard C

    2010-07-01

    Primary Epstein-Barr virus (EBV) infection often results in infectious mononucleosis and is associated with serious sequelae. No treatment is approved for EBV infection, and an antiviral intervention would be significant. The objectives of this study are to characterize the pharmacokinetics and explore the pharmacodynamics of acyclovir in plasma and oral washings of 8 subjects receiving 7 days of valacyclovir 1500 mg twice daily for EBV infectious mononucleosis. Virologic and clinical responses are assessed over 12 days. Acyclovir is measured by liquid chromatography/ultraviolet detection. EBV DNA is quantitated by TaqMan polymerase chain reaction. NONMEM VI and linear regression are used for data analysis. Acyclovir profiles in plasma and oral washings are consistent with a 1-compartment model. Final model estimates of clearance, volume of distribution, and fraction of acyclovir in oral wash supernatant are 49.9 L/h, 74.1 L, and 1.14%, respectively. The quantity of EBV DNA in oral washings and blood, and the severity of illness, measured by a graded scale, decrease during treatment. After treatment, viral rebound occurs in oral washings but not in blood, and the severity of illness continues to decline. Acyclovir pharmacokinetic parameters do not correlate with response metrics. These results support further studies of valacyclovir for EBV infectious mononucleosis.

  16. A prospective clinical study of Epstein-Barr virus and host interactions during acute infectious mononucleosis.

    Science.gov (United States)

    Balfour, Henry H; Holman, Carol J; Hokanson, Kristin M; Lelonek, Meghan M; Giesbrecht, Jill E; White, Dana R; Schmeling, David O; Webb, Chiu-Ho; Cavert, Winston; Wang, David H; Brundage, Richard C

    2005-11-01

    Characterizing virus-host interactions during self-limited infectious mononucleosis could explain how Epstein-Barr virus (EBV) replication is normally controlled and provide insight into why certain immunocompromised patients fail to contain it. University students had an average of 7 clinical and virologic evaluations during acute infectious mononucleosis. EBV was quantified in 697 samples of oral wash fluid, whole blood, peripheral blood mononuclear cells (PBMCs), and plasma by a real-time (TaqMan) polymerase chain reaction (qEBV) assay developed in our laboratory. Twenty of 25 subjects had serologically confirmed primary EBV infection. EBV was cleared from whole blood by a first-order process with a median half-life of 3 days, and its quantity was associated with severity of illness (r2=0.82). Oral shedding persisted at a median of >or=1x104 copies/mL for 32 weeks and was unrelated to severity of illness. Subjects with nonprimary EBV infection shed virus intermittently, and median quantities for all samples became undetectable within 4 weeks. Using a novel qEBV assay, we demonstrated that young adults with primary EBV infection rapidly cleared virus from blood but not from the oropharynx. High oral concentrations of EBV in asymptomatic persons who have resumed normal activities support the concept that infectious mononucleosis is most likely acquired by kissing.

  17. Study of the association between 3111T/C polymorphism of the CLOCK gene and the presence of overweight in schoolchildren,

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    Nayara P. Giovaninni

    2014-09-01

    Full Text Available Objectives: To evaluate the association between 3111T/C polymorphism of the CLOCK gene and the presence of obesity and sleep duration in children aged 6-13 years. In adults, this genetic variant has been associated with duration of sleep, ghrelin levels, weight, and eating habits. Although short sleep duration has been linked to obesity in children, no study has aimed to identify the possible molecular mechanisms of this association to date. Methods: Weight, height, and circumferences were transformed into Z-scores for age and gender. Genotyping was performed using TaqMan methodology. A questionnaire regarding hours of sleep was provided to parents. The appropriate statistical tests were performed. Results: This study evaluated 370 children (45% males, 55% females, mean age 8.5 ± 1.5 years. The prevalence of overweight was 18%. The duration of sleep was, on average, 9.7 hours, and was inversely related to age (p < 0.001. Genotype distribution was: 4% CC, 31% CT, and 65% TT. There was a trend toward higher prevalence of overweight in children who slept less than nine hours (23% when compared to those who slept more than ten hours (16%, p = 0.06. Genotype was not significantly correlated to any of the assessed outcomes. Conclusions: The CLOCK 3111T/C polymorphism was not significantly associated with overweight or sleep duration in children in this city.

  18. Association study of ghrelin receptor gene polymorphisms in rheumatoid arthritis.

    Science.gov (United States)

    Robledo, G; Rueda, B; Gonzalez-Gay, M A; Fernández, B; Lamas, J R; Balsa, A; Pascual-Salcedo, D; García, A; Raya, E; Martín, J

    2010-01-01

    Ghrelin is a newly characterised growth hormone (GH) releasing peptide widely distributed that may play an important role in the regulation of metabolic balance in inflammatory diseases such as rheumatoid arthritis (RA) by decreasing the pro-inflammatory Th1 responses. In this study we investigated the possible contribution of several polymorphisms in the functional Ghrelin receptor to RA susceptibility. A screening of 3 single nucleotide polymorphisms (SNPs) was performed in a total of 950 RA patients and 990 healthy controls of Spanish Caucasian origin. Genotyping of all 3 SNPs was performed by real-time polymerase chain reaction technology, using the TaqMan 5'-allele discrimination assay. We observed no statistically significant deviation between RA patients and controls for the GHSR SNPs analysed. In addition, we performed a haplotype analysis that did not reveal an association with RA susceptibility. The stratification analysis for the presence of shared epitope (SE), rheumatoid factor (RF) or antibodies anti cyclic citrullinated peptide (anti-CCP) did not detect significant association of the GHSR polymorphisms with RA. These findings suggest that the GHSR gene polymorphisms do not appear to play a major role in RA genetic predisposition in our population.

  19. Lack of association of ghrelin precursor gene variants and percentage body fat or serum lipid profiles.

    Science.gov (United States)

    Martin, Glynn R; Loredo, J C; Sun, Guang

    2008-04-01

    Ghrelin has been recognized for its involvement in food intake, control of energy homeostasis, and lipid metabolism. However, the roles of genetic variations in the ghrelin precursor gene (GHRL) on body compositions and serum lipids are not clear in humans. Our study investigated five single-nucleotide polymorphisms (SNPs) within GHRL to determine their relationship with body fat percentage (BF), trunk fat percentage (TF), lower body (legs) fat percentage (LF), and serum lipids in 1,464 subjects, which were recruited from the genetically homogeneous population of Newfoundland and Labrador (NL), Canada. Serum glucose, insulin, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and triglycerides were determined. Five SNPs are rs35684 (A/G: a transition substitution in exon 1), rs4684677 (A/T: a missense mutation), rs2075356 (C/T: intron), rs26802 (G/T: intron), and rs26311 (A/G: near the 3' untranslated region) of GHRL were genotyped using TaqMan validated or functionally tested SNP genotyping assays. Our study found no significant evidence of an allele or genotype association between any of the variant sites and body compositions or serum lipids. Furthermore, haplotype frequencies were not found to be significantly different between lean and obese subjects. In summary, the results of our study do not support a significant role for genetic variations in GHRL in the differences of body fat and serum lipid profiles in the NL population.

  20. Study of the association between 3111T/C polymorphism of the CLOCK gene and the presence of overweight in schoolchildren.

    Science.gov (United States)

    Giovaninni, Nayara P; Fuly, Jeanne T; Moraes, Leonardo I; Coutinho, Thais N; Trarbach, Ericka B; Jorge, Alexander A de L; Costalonga, Everlayny F

    2014-01-01

    To evaluate the association between 3111T/C polymorphism of the CLOCK gene and the presence of obesity and sleep duration in children aged 6-13 years. In adults, this genetic variant has been associated with duration of sleep, ghrelin levels, weight, and eating habits. Although short sleep duration has been linked to obesity in children, no study has aimed to identify the possible molecular mechanisms of this association to date. Weight, height, and circumferences were transformed into Z-scores for age and gender. Genotyping was performed using TaqMan methodology. A questionnaire regarding hours of sleep was provided to parents. The appropriate statistical tests were performed. This study evaluated 370 children (45% males, 55% females, mean age 8.5 ± 1.5 years). The prevalence of overweight was 18%. The duration of sleep was, on average, 9.7hours, and was inversely related to age (ppolymorphism was not significantly associated with overweight or sleep duration in children in this city. Copyright © 2014 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  1. Genetic association between ghrelin polymorphisms and Alzheimer's disease in a Japanese population.

    Science.gov (United States)

    Shibata, Nobuto; Ohnuma, Tohru; Kuerban, Bolati; Komatsu, Miwa; Arai, Heii

    2011-01-01

    Ghrelin has been reported to enter the hippocampus and to bind to the neurons of the hippocampal formation. This peptide also affects neuronal glucose uptake and decreases tau hyperphosphorylation. There is increasing evidence suggesting an association between ghrelin and Alzheimer's disease (AD) pathology. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) of the ghrelin gene are associated with AD. The SNPs were genotyped using TaqMan technology and were analyzed using a case-control study design. Our case-control dataset consisted of 182 AD patients and 143 age-matched controls. Hardy-Weinberg equilibrium and linkage disequilibrium analyses suggest that the region in and around the gene is highly polymorphic. One SNP, rs4684677 (Leu90Gln), showed a marginal association with age of AD onset. We did not detect any association between the other SNPs of the ghrelin gene and AD. There have been few genetic studies on the relationship between circulating ghrelin and functional SNPs. Further multifactorial studies are needed to clarify the relationship between ghrelin and AD. Copyright © 2011 S. Karger AG, Basel.

  2. A Genomics-Based Model for Prediction of Severe Bioprosthetic Mitral Valve Calcification.

    Science.gov (United States)

    Ponasenko, Anastasia V; Khutornaya, Maria V; Kutikhin, Anton G; Rutkovskaya, Natalia V; Tsepokina, Anna V; Kondyukova, Natalia V; Yuzhalin, Arseniy E; Barbarash, Leonid S

    2016-08-31

    Severe bioprosthetic mitral valve calcification is a significant problem in cardiovascular surgery. Unfortunately, clinical markers did not demonstrate efficacy in prediction of severe bioprosthetic mitral valve calcification. Here, we examined whether a genomics-based approach is efficient in predicting the risk of severe bioprosthetic mitral valve calcification. A total of 124 consecutive Russian patients who underwent mitral valve replacement surgery were recruited. We investigated the associations of the inherited variation in innate immunity, lipid metabolism and calcium metabolism genes with severe bioprosthetic mitral valve calcification. Genotyping was conducted utilizing the TaqMan assay. Eight gene polymorphisms were significantly associated with severe bioprosthetic mitral valve calcification and were therefore included into stepwise logistic regression which identified male gender, the T/T genotype of the rs3775073 polymorphism within the TLR6 gene, the C/T genotype of the rs2229238 polymorphism within the IL6R gene, and the A/A genotype of the rs10455872 polymorphism within the LPA gene as independent predictors of severe bioprosthetic mitral valve calcification. The developed genomics-based model had fair predictive value with area under the receiver operating characteristic (ROC) curve of 0.73. In conclusion, our genomics-based approach is efficient for the prediction of severe bioprosthetic mitral valve calcification.

  3. Identification of the PLA2G6 c.1579G>A Missense Mutation in Papillon Dog Neuroaxonal Dystrophy Using Whole Exome Sequencing Analysis.

    Directory of Open Access Journals (Sweden)

    Masaya Tsuboi

    Full Text Available Whole exome sequencing (WES has become a common tool for identifying genetic causes of human inherited disorders, and it has also recently been applied to canine genome research. We conducted WES analysis of neuroaxonal dystrophy (NAD, a neurodegenerative disease that sporadically occurs worldwide in Papillon dogs. The disease is considered an autosomal recessive monogenic disease, which is histopathologically characterized by severe axonal swelling, known as "spheroids," throughout the nervous system. By sequencing all eleven DNA samples from one NAD-affected Papillon dog and her parents, two unrelated NAD-affected Papillon dogs, and six unaffected control Papillon dogs, we identified 10 candidate mutations. Among them, three candidates were determined to be "deleterious" by in silico pathogenesis evaluation. By subsequent massive screening by TaqMan genotyping analysis, only the PLA2G6 c.1579G>A mutation had an association with the presence or absence of the disease, suggesting that it may be a causal mutation of canine NAD. As a human homologue of this gene is a causative gene for infantile neuroaxonal dystrophy, this canine phenotype may serve as a good animal model for human disease. The results of this study also indicate that WES analysis is a powerful tool for exploring canine hereditary diseases, especially in rare monogenic hereditary diseases.

  4. Lack of association between miR-218 rs11134527 A>G and Kawasaki disease susceptibility.

    Science.gov (United States)

    Pi, Lei; Fu, Lanyan; Xu, Yufen; Che, Di; Deng, Qiulian; Huang, Xijing; Li, Meiai; Zhang, Li; Huang, Ping; Gu, Xiaoqiong

    2018-05-01

    Abstract Kawasaki disease (KD) is a type of disease that includes the development of a fever that lasts at least five days and involves the clinical manifestation of multicellular vasculitis. KD has become one of the most common pediatric cardiovascular diseases. Previous studies have reported that miR-218 rs11134527 A>G is associated with susceptibility to various cancer risks. However, there is a lack of evidence regarding the relationship between this polymorphism and KD risk. This study explored the correlation between the miR-218 rs11134527 A>G polymorphism and the risk of KD. We recruited 532 patients with KD and 623 controls to genotype the miR-218 rs11134527 A>G polymorphism with a TaqMan allelic discrimination assay. Our results illustrated that the miR-218 rs11134527 A>G polymorphism was not associated with KD risk. In an analysis stratified by age, sex, and coronary artery lesions, we found only that the risk of KD was significantly decreased for children older than 5 years (GG vs. AA/AG: adjusted OR=0.26, 95% CI=0.07-0.94, P =0.041). This study demonstrated that the miR-218 rs1113452 A>G polymorphism may have an age-related relationship with KD susceptibility that has not previously been revealed. ©2018 The Author(s).

  5. Macrophage Migration Inhibitory Factor (MIF) Gene Promotor Polymorphism Is Associated with Increased Fibrosis in Biliary Atresia Patients, but Not with Disease Susceptibility.

    Science.gov (United States)

    Sadek, Khaled H; Ezzat, Sameera; Abdel-Aziz, Samira A; Alaraby, Hanaa; Mosbeh, Asmaa; Abdel-Rahman, Mohamed H

    2017-09-01

    Two polymorphisms, rs755622 and rs5844572, in the promoter region of the macrophage migration inhibitory factor (MIF) gene influence the basal and/or induced transcriptional activity and have been linked to several inflammatory and autoimmune diseases. The aim of this study was to investigate the association between these two polymorphisms and disease susceptibility in patients with biliary atresia (BA). Allele frequencies of rs755622 and rs5844572 were assessed in 60 Egyptian infants with a confirmed diagnosis of BA. DNA was extracted from archival material. For the rs755622, samples were tested using Taqman real-time PCR, and for the rs5844572, samples were tested using fluorescence-based genotyping. The allele frequency in the general population was assessed in 141 healthy adults from the same geographical location. No statistical differences were observed in the allele frequencies of either rs755622 or rs5844572 between BA patients and controls. The homozygous and heterozygous short repeats (5/5, or 5/X) of rs5844572 were observed more frequently (16/28, 57.1%) in BA patients with mild to moderate fibrosis compared with those with marked fibrosis (10/32, 31.3%). The difference was statistically significant (P  =  0.032). In conclusion, we observed no association between MIF rs755622 and rs5844572 polymorphisms and susceptibility to BA; however, the rs5844572 could be linked to the rate of progression of the disease and extent of fibrosis. © 2017 John Wiley & Sons Ltd/University College London.

  6. Association of HMOX1 and NQO1 Polymorphisms with Metabolic Syndrome Components.

    Science.gov (United States)

    Martínez-Hernández, Angélica; Córdova, Emilio J; Rosillo-Salazar, Oscar; García-Ortíz, Humberto; Contreras-Cubas, Cecilia; Islas-Andrade, Sergio; Revilla-Monsalve, Cristina; Salas-Labadía, Consuelo; Orozco, Lorena

    2015-01-01

    Metabolic syndrome (MetS) is among the most important public health problems worldwide, and is recognized as a major risk factor for various illnesses, including type 2 diabetes mellitus, obesity, and cardiovascular diseases. Recently, oxidative stress has been suggested as part of MetS aetiology. The heme oxygenase 1 (HMOX1) and NADH:quinone oxidoreductase 1 (NQO1) genes are crucial mediators of cellular defence against oxidative stress. In the present study, we analysed the associations of HMOX1 (GT)n and NQO1 C609T polymorphisms with MetS and its components. Our study population comprised 735 Mexican Mestizos unrelated volunteers recruited from different tertiary health institutions from Mexico City. In order to know the HMOX1 (GT)n and NQO1 C609T allele frequencies in Amerindians, we included a population of 241 Amerindian native speakers. Their clinical and demographic data were recorded. The HMOX1 (GT)n polymorphism was genotyped using PCR and fluorescence technology. NQO1 C609T polymorphism genotyping was performed using TaqMan probes. Short allele (metabolic disorders, including high systolic and diastolic blood pressure, hypertriglyceridemia, and low HDL-c levels in Mexican Mestizo individuals.

  7. Comprehensive analysis of three TYK2 gene variants in the susceptibility to Chagas disease infection and cardiomyopathy

    Science.gov (United States)

    Carmona, F. David; Dolade, Nuria; Vargas, Sofia; Echeverría, Luis Eduardo; González, Clara Isabel; Martin, Javier

    2018-01-01

    Tyrosine kinase 2 (TYK2) is a member of the Janus kinases family implicated in the signal transduction of type I interferons and several interleukins. It has been described that genetic mutations within TYK2 lead to multiple deleterious effects in the immune response. In this work, we have analyzed three functional independent variants from the frequency spectrum on the TYK2 gene (common and low-frequency variants) suggested to reduce the function of the gene in mediating cytokine signaling and the susceptibility to infections by Trypanosoma cruzi and/or the development of Chagas cardiomyopathy in the Colombian population. A total of 1,323 individuals from a Colombian endemic region for Chagas disease were enrolled in the study. They were classified as seronegative (n = 445), seropositive asymptomatic (n = 336), and chronic Chagas Cardiomyopathy subjects (n = 542). DNA samples were genotyped using TaqMan probes. Our results showed no statistically significant differences between the allelic frequencies of the three analyzed variants when seropositive and seronegative individuals were compared, therefore these variants were not associated with susceptibility to Chagas disease. Moreover, when Chagas cardiomyopathy patients were compared to asymptomatic patients, no significant associations were found. Previous reports highlighted the association of this gene in immune-related disorders under an autoimmunity context, but not predisposing patients to infectious diseases, which is consistent with our findings. Therefore, according to our results, TYK2 gene variants do not seem to play an important role in Chagas disease susceptibility and/or chronic Chagas cardiomyopathy. PMID:29304122

  8. New PCR diagnostic systems for the detection and quantification of porcine cytomegalovirus (PCMV).

    Science.gov (United States)

    Morozov, Vladimir A; Morozov, Alexey V; Denner, Joachim

    2016-05-01

    Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants.

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

    Science.gov (United States)

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

    2017-10-03

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

  10. Association of OCT derived drusen measurements with AMD associated-genotypic SNPs in Amish population.

    Science.gov (United States)

    Chavali, Venkata Ramana Murthy; Diniz, Bruno; Huang, Jiayan; Ying, Gui-Shuang; Sadda, SriniVas R; Stambolian, Dwight

    To investigate the association of OCT derived drusen measures in Amish age-related macular degeneration (AMD) patients with known loci for macular degeneration. Members of the Old Order Amish community in Pennsylvania ages 50 and older were assessed for drusen area, volume and regions of retinal pigment epithelium (RPE) atrophy using a Cirrus High- Definition-OCT. Measurements were obtained in the macula region within a central circle (CC) of 3 mm diameter and a surrounding perifoveal ring (PR) of 3 to 5 mm diameter using the Cirrus OCT RPE analysis software. Other demographic information including age, gender and smoking status were collected. Study subjects were further genotyped to determine their risk for the AMD associated SNPs in SYN3, LIPC, ARMS2, C3, CFB, CETP, CFI and CFH genes using TaqMan genotyping assays. The association of genotypes with OCT measures were assessed using linear trend p-values calculated from univariate and multivariate generalized linear models. 432 eyes were included in the analysis. Multivariate analysis (adjusted by age, gender and smoking status) confirmed the known significant association between AMD and macular drusen with the number of CFH risk alleles for drusen area (area increased 0.12 mm 2 for a risk allele increase, pAmish AMD population.

  11. Interaction between VEGF receptor-2 gene polymorphisms and dietary patterns on blood glucose and lipid levels in Chinese Malaysian adults.

    Science.gov (United States)

    Yap, Roseline Wai Kuan; Shidoji, Yoshihiro; Hon, Wei Min; Masaki, Motofumi

    2011-01-01

    The prevalence of lifestyle-related chronic diseases is increasing and gene-diet interaction studies are limited among the Malaysian population. This study was conducted to evaluate the association and interaction effects of vascular endothelial growth factor receptor-2(VEGFR2) gene polymorphisms and dietary patterns on anthropometric and biochemical risk factors of chronic diseases in 179 Chinese Malaysian adults. Genotyping of rs1870377 and rs2071559 was performed by real-time PCR using TaqMan probes. Dietary patterns were constructed from the food frequency questionnaire using factor analysis. Anthropometric measurements: body mass index (BMI), systolic and diastolic blood pressure and biomarkers: blood glucose, glycated hemoglobin A1c (HbA1c) and lipids were obtained. Two dietary patterns: 'Balanced diet' and 'Meat, rice and noodles diet' (MRND) were extracted. MRND was associated with higher BMI, blood pressure, blood glucose and lipids, while T alleles in both rs1870377 and rs2071559 were associated with higher blood lipids (p Malaysian adults. Copyright © 2012 S. Karger AG, Basel.

  12. Association and Interaction Effect of AGTR1 and AGTR2 Gene Polymorphisms with Dietary Pattern on Metabolic Risk Factors of Cardiovascular Disease in Malaysian Adults.

    Science.gov (United States)

    Yap, Roseline Wai Kuan; Shidoji, Yoshihiro; Yap, Wai Sum; Masaki, Motofumi

    2017-08-09

    Gene-diet interaction using a multifactorial approach is preferred to study the multiple risk factors of cardiovascular disease (CVD). This study examined the association and gene-diet interaction effects of the angiotensin II type 1 receptor ( AGTR1 ) gene (rs5186), and type 2 receptor ( AGTR2 ) gene (rs1403543) polymorphisms on metabolic risk factors of CVD in Malaysian adults. CVD parameters (BMI, blood pressure, glycated hemoglobin, total cholesterol (TC), triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol (HDL-C), and TC/HDL-C ratio), and constructed dietary patterns "vegetables, fruits, and soy diet" (VFSD), and "rice, egg, and fish diet" (REFD) were obtained from previous studies. Genotyping analysis was performed by real-time PCR using Taqman probes. The subjects were 507 adults (151 Malays; 179 Chinese; and 177 Indians). Significant genetic associations were obtained on blood lipids for rs5186 in Malays and Chinese, and rs1403543 in Chinese females. The significant gene-diet interaction effects after adjusting for potential confounders were: rs5186 × VFSD on blood pressure in Malays ( p = 0.016), and in Chinese on blood lipids for rs5186 × REFD ( p = 0.009-0.023), and rs1403543 × VFSD in female subjects ( p = 0.001-0.011). Malays and Chinese showed higher risk for blood pressure and/or lipids involving rs5186 and rs1403543 SNPs together with gene-diet interactions, but not Indians.

  13. Molecular Endoscopic Ultrasound for Diagnosis of Pancreatic Cancer

    International Nuclear Information System (INIS)

    Bournet, Barbara; Pointreau, Adeline; Delpu, Yannick; Selves, Janick; Torrisani, Jerome; Buscail, Louis; Cordelier, Pierre

    2011-01-01

    Endoscopic ultrasound-guided fine needle aspiration-biopsy is a safe and effective technique in diagnosing and staging of pancreatic ductal adenocarcinoma. However its predictive negative value does not exceed 50% to 60%. Unfortunately, the majority of pancreatic cancer patients have a metastatic and/or a locally advanced disease (i.e., not eligible for curative resection) which explains the limited access to pancreatic tissue specimens. Endoscopic ultrasound-guided fine needle aspiration-biopsy is the most widely used approach for cytological and histological material sampling in these situations used in up to two thirds of patients with pancreatic cancer. Based on this unique material, we and others developed strategies to improve the differential diagnosis between carcinoma and inflammatory pancreatic lesions by analysis of KRAS oncogene mutation, microRNA expression and methylation, as well as mRNA expression using both qRT-PCR and Low Density Array Taqman analysis. Indeed, differentiating pancreatic cancer from pseudotumoral chronic pancreatitis remains very difficult in current clinical practice, and endoscopic ultrasound-guided fine needle aspiration-biopsy analysis proved to be very helpful. In this review, we will compile the clinical and molecular advantages of using endoscopic ultrasound-guided fine needle aspiration-biopsy in managing pancreatic cancer

  14. Lack of Association Between Polymorphisms in Dopa Decarboxylase and Dopamine Receptor-1 Genes With Childhood Autism in Chinese Han Population.

    Science.gov (United States)

    Yu, Hong; Liu, Jun; Yang, Aiping; Yang, Guohui; Yang, Wenjun; Lei, Heyue; Quan, Jianjun; Zhang, Zengyu

    2016-04-01

    Genetic factors play an important role in childhood autism. This study is to determine the association of single-nucleotide polymorphisms in dopa decarboxylase (DDC) and dopamine receptor-1 (DRD1) genes with childhood autism, in a Chinese Han population. A total of 211 autistic children and 250 age- and gender-matched healthy controls were recruited. The severity of disease was determined by Children Autism Rating Scale scores. TaqMan Probe by real-time polymerase chain reaction was used to determine genotypes and allele frequencies of single-nucleotide polymorphism rs6592961 in DDC and rs251937 in DRD1. Case-control and case-only studies were respectively performed, to determine the contribution of both single-nucleotide polymorphisms to the predisposition of disease and its severity. Our results showed that there was no significant association of the genotypes and allele frequencies of both single-nucleotide polymorphisms concerning childhood autism and its severity. More studies with larger samples are needed to corroborate their predicting roles. © The Author(s) 2015.

  15. The Genetic Link between Parkinson's Disease and the Kynurenine Pathway Is Still Missing.

    Science.gov (United States)

    Török, Nóra; Török, Rita; Szolnoki, Zoltán; Somogyvári, Ferenc; Klivényi, Péter; Vécsei, László

    2015-01-01

    Background. There is substantial evidence that the kynurenine pathway (KP) plays a role in the normal physiology of the brain and is involved in the pathology of neurodegenerative disorders such as Huntington's disease and Parkinson's disease (PD). Objective. We set out to investigate the potential roles in PD of single nucleotide polymorphisms (SNPs) from one of the key enzymes of the KP, kynurenine 3-monooxygenase (KMO). Methods. 105 unrelated, clinically definitive PD patients and 131 healthy controls were enrolled to investigate the possible effects of the different alleles of KMO. Fluorescently labeled TaqMan probes were used for allele discrimination. Results. None of the four investigated SNPs proved to be associated with PD or influenced the age at onset of the disease. Conclusions. The genetic link between the KP and PD is still missing. The investigated SNPs presumably do not appear to influence the function of KMO and probably do not contain binding sites for regulatory proteins of relevance in PD. This is the first study to assess the genetic background behind the biochemical alterations of the kynurenine pathway in PD, directing the attention to this previously unexamined field.

  16. The Genetic Link between Parkinson’s Disease and the Kynurenine Pathway Is Still Missing

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    Nóra Török

    2015-01-01

    Full Text Available Background. There is substantial evidence that the kynurenine pathway (KP plays a role in the normal physiology of the brain and is involved in the pathology of neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease (PD. Objective. We set out to investigate the potential roles in PD of single nucleotide polymorphisms (SNPs from one of the key enzymes of the KP, kynurenine 3-monooxygenase (KMO. Methods. 105 unrelated, clinically definitive PD patients and 131 healthy controls were enrolled to investigate the possible effects of the different alleles of KMO. Fluorescently labeled TaqMan probes were used for allele discrimination. Results. None of the four investigated SNPs proved to be associated with PD or influenced the age at onset of the disease. Conclusions. The genetic link between the KP and PD is still missing. The investigated SNPs presumably do not appear to influence the function of KMO and probably do not contain binding sites for regulatory proteins of relevance in PD. This is the first study to assess the genetic background behind the biochemical alterations of the kynurenine pathway in PD, directing the attention to this previously unexamined field.

  17. Feasibility of using tissue microarray cores of paraffin-embedded breast cancer tissue for measurement of gene expression: a proof-of-concept study.

    Science.gov (United States)

    Drury, Suzanne; Salter, Janine; Baehner, Frederick L; Shak, Steven; Dowsett, Mitch

    2010-06-01

    To determine whether 0.6 mm cores of formalin-fixed paraffin-embedded (FFPE) tissue, as commonly used to construct immunohistochemical tissue microarrays, may be a valid alternative to tissue sections as source material for quantitative real-time PCR-based transcriptional profiling of breast cancer. Four matched 0.6 mm cores of invasive breast tumour and two 10 microm whole sections were taken from eight FFPE blocks. RNA was extracted and reverse transcribed, and TaqMan assays were performed on the 21 genes of the Oncotype DX Breast Cancer assay. Expression of the 16 recurrence-related genes was normalised to the set of five reference genes, and the recurrence score (RS) was calculated. RNA yield was lower from 0.6 mm cores than from 10 microm whole sections, but was still more than sufficient to perform the assay. RS and single gene data from cores were highly comparable with those from whole sections (RS p=0.005). Greater variability was seen between cores than between sections. FFPE sections are preferable to 0.6 mm cores for RNA profiling in order to maximise RNA yield and to allow for standard histopathological assessment. However, 0.6 mm cores are sufficient and would be appropriate to use for large cohort studies.

  18. Paraoxonase1 Genetic Polymorphisms in a Mixed Ancestry African Population

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    M. Macharia

    2014-01-01

    Full Text Available Paraoxonase 1 (PON1 activity is markedly influenced by coding polymorphisms, Q/R at position 192 and M/L at position 55 of the PON1 gene. We investigated the frequencies of these polymorphisms and their effects on PON1 and antioxidant activities in 844 South African mixed ancestry individuals. Genotyping was done using allele-specific TaqMan technology, PON1 activities were measured using paraoxon and phenylacetate, oxidative status was determined by measuring the antioxidant activities of ferric reducing antioxidant power and trolox equivalent antioxidant capacity, and lipid peroxidation markers included malondialdehyde and oxidized LDL. The frequencies of Q192R and L55M were 47.6% and 28.8%, respectively, and the most common corresponding alleles were 192R (60.4% and 55M (82.6%. The Q192 was significantly associated with 5.8 units’ increase in PON1 concentration and 15.4 units’ decrease in PONase activity after adjustment for age, sex, BMI, and diabetes, with suggestion of differential effects by diabetes status. The PON1 L55 variant was associated with none of the measured indices. In conclusion, we have shown that the Q192R polymorphism is a determinant of both PON1 concentration and activity and this association appeared to be enhanced in subjects with diabetes.

  19. Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR.

    Science.gov (United States)

    Espiñeira, Montserrat; Vieites, Juan M

    2012-12-15

    The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay. The method can be applied to all kinds of products; fresh, frozen and processed, including those undergoing intensive processes of transformation. This methodology was validated to check how the degree of food processing affects the method and the detection of each species. Moreover, it was applied to 34 commercial samples to evaluate the labeling of products made from them. The methodology herein developed is useful to check the fulfillment of labeling regulations for seafood products and to verify traceability in commercial trade and for fisheries control. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. [Distribution of three polymorphisms of the TSLP gen in African-descendent population from San Basilio de Palenque, Colombia].

    Science.gov (United States)

    Fang, Luis; Martínez, Beatriz; Marrugo, Javier

    2013-01-01

    Thymic stromal lymphopoietin (TSLP) has been linked as a susceptibility gene for the development of allergic diseases. It is known that the population of Cartagena is a triethnic mix, in which the component of African ancestry was significantly associated with risk of asthma and high total serum IgE levels. This component comes from African slaves brought into the continent and settled in "palenques", one of them is San Basilio de Palenque, in the Colombian Caribbean Coast. To analyze the distribution of single nucleotide polymorphisms (SNP) rs1837253, rs17551370 and rs2289276 located in TSLP gene, in the African-descendent population of San Basilio de Palenque. By real time-PCR and probes TaqMan SNP Genotyping™, we genotyped three polymorphisms in 80 individuals of African-descent aged 5 to 18 years of age. The frequency of the rs1837253 allele T was 41.9%, for the allele A, 14.3% for rs17551370, and 22.5% for the allele T of rs2289276. The rs17551370 and rs2289276 distribution remained in Hardy- Weinberg genetic equilibrium. The allele frequency of each SNP did not show statistically significant differences with those reported for other African and African-descendent populations. The three polymorphisms in the TSLP were present in the sample population of San Basilio de Palenque and its distribution is similar to that reported for African populations and African ancestry in America.

  1. No evidence of XMRV or related retroviruses in a London HIV-1-positive patient cohort.

    Directory of Open Access Journals (Sweden)

    Eleanor R Gray

    2011-03-01

    Full Text Available Several studies have implicated a recently discovered gammaretrovirus, XMRV (Xenotropic murine leukaemia virus-related virus, in chronic fatigue syndrome and prostate cancer, though whether as causative agent or opportunistic infection is unclear. It has also been suggested that the virus can be found circulating amongst the general population. The discovery has been controversial, with conflicting results from attempts to reproduce the original studies.We extracted peripheral blood DNA from a cohort of 540 HIV-1-positive patients (approximately 20% of whom have never been on anti-retroviral treatment and determined the presence of XMRV and related viruses using TaqMan PCR. While we were able to amplify as few as 5 copies of positive control DNA, we did not find any positive samples in the patient cohort.In view of these negative findings in this highly susceptible group, we conclude that it is unlikely that XMRV or related viruses are circulating at a significant level, if at all, in HIV-1-positive patients in London or in the general population.

  2. Correlation of HAMP gene polymorphisms and expression with the susceptibility and length of hospital stays in Taiwanese children with Kawasaki disease

    Science.gov (United States)

    Lu, Hsing-Fang; Wong, Henry Sung-Ching; Yu, Hong-Ren; Kuo, Hsing-Chun; Huang, Fu-Chen; Lo, Mao-Hung; Hsieh, Kai-Sheng; Chen, Su-Fen; Chang, Wei-Chiao; Kuo, Ho-Chang

    2017-01-01

    Kawasaki disease (KD) is a form of systemic vasculitis. Regarding its pathogenesis, HAMP gene encoding hepcidin, which is significant for iron metabolism, has a vital function. In this study, we recruited a total of 381 KD patients for genotyping. Data from 997 subjects (500 subjects from cohort 1; 497 subjects from cohort 2) were used for analysis. Using TaqMan allelic discrimination, we determined five tag SNPs (rs916145, rs10421768, rs3817623, rs7251432, and rs2293689). Treatment outcome data related to such clinical phenotypes as coronary artery lesions (CAL), coronary artery aneurysms (CAA), and intravenous immunoglobulin (IVIG) effects were also collected. Furthermore, we measured plasma hepcidin levels with an enzyme-linked immunosorbent assay. We found that HAMP gene polymorphism (rs7251432, and rs2293689) was significantly correlated with KD risk and that plasma hepcidin levels both before and after IVIG treatment had a significantly positive correlation with length of hospital stays (R = 0.217, p = 0.046 and R = 0.381, p < 0.0001, respectively). In contrast, plasma hepcidin levels has a negative correlation with KD patients’ albumin levels (R = −0.27, p < 0.001) prior to IVIG treatment. This study's findings indicate that HAMP might have a role in the disease susceptibility, as well as its expressions correlated length of hospital stays, and albumin levels in Taiwanese children with KD. PMID:28881695

  3. Genetic variants of CD209 associated with Kawasaki disease susceptibility.

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    Ho-Chang Kuo

    Full Text Available BACKGROUND: Kawasaki disease (KD is a systemic vasculitis with unknown etiology mainly affecting children in Asian countries. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209 in humans was showed to trigger an anti-inflammatory cascade and associated with KD susceptibility. This study was conducted to investigate the association between genetic polymorphisms of CD209 and the risk KD. METHODS: A total of 948 subjects (381 KD and 567 controls were recruited. Nine tagging SNPs (rs8112310, rs4804800, rs11465421, rs1544766, rs4804801, rs2287886, rs735239, rs735240, rs4804804 were selected for TaqMan allelic discrimination assay. Clinical phenotypes, coronary artery lesions (CAL and intravenous immunoglobulin (IVIG treatment outcomes were collected for analysis. RESULTS: Significant associations were found between CD209 polymorphisms (rs4804800, rs2287886, rs735240 and the risk of KD. Haplotype analysis for CD209 polymorphisms showed that A/A/G haplotype (P = 0.0002, OR = 1.61 and G/A/G haplotype (P = 0.0365, OR = 1.52 had higher risk of KD as compared with G/G/A haplotype in rs2287886/rs735239/rs735240 pairwise allele analysis. There were no significant association in KD with regards to CAL formation and IVIG treatment responses. CONCLUSION: CD209 polymorphisms were responsible for the susceptibility of KD, but not CAL formation and IVIG treatment responsiveness.

  4. Genetic Variants of CD209 Associated with Kawasaki Disease Susceptibility

    Science.gov (United States)

    Kuo, Ho-Chang; Huang, Ying-Hsien; Chien, Shu-Chen; Yu, Hong-Ren; Hsieh, Kai-Sheng; Hsu, Yu-Wen; Chang, Wei-Chiao

    2014-01-01

    Background Kawasaki disease (KD) is a systemic vasculitis with unknown etiology mainly affecting children in Asian countries. Dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209) in humans was showed to trigger an anti-inflammatory cascade and associated with KD susceptibility. This study was conducted to investigate the association between genetic polymorphisms of CD209 and the risk KD. Methods A total of 948 subjects (381 KD and 567 controls) were recruited. Nine tagging SNPs (rs8112310, rs4804800, rs11465421, rs1544766, rs4804801, rs2287886, rs735239, rs735240, rs4804804) were selected for TaqMan allelic discrimination assay. Clinical phenotypes, coronary artery lesions (CAL) and intravenous immunoglobulin (IVIG) treatment outcomes were collected for analysis. Results Significant associations were found between CD209 polymorphisms (rs4804800, rs2287886, rs735240) and the risk of KD. Haplotype analysis for CD209 polymorphisms showed that A/A/G haplotype (P = 0.0002, OR = 1.61) and G/A/G haplotype (P = 0.0365, OR = 1.52) had higher risk of KD as compared with G/G/A haplotype in rs2287886/rs735239/rs735240 pairwise allele analysis. There were no significant association in KD with regards to CAL formation and IVIG treatment responses. Conclusion CD209 polymorphisms were responsible for the susceptibility of KD, but not CAL formation and IVIG treatment responsiveness. PMID:25148534

  5. Integrated Genomic Analysis of Sézary Syndrome

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    Xin Mao

    2011-01-01

    Full Text Available Sézary syndrome (SS is a rare variant of primary cutaneous T-cell lymphoma. Little is known about the underlying pathogenesis of S. To address this issue, we used Affymetrix 10K SNP microarray to analyse 13 DNA samples isolated from 8 SS patients and qPCR with ABI TaqMan SNP genotyping assays for the validation of the SNP microarray results. In addition, we tested the impact of SNP loss of heterozygosity (LOH identified in SS cases on the gene expression profiles of SS cases detected with Affymetrix GeneChip U133A. The results showed: (1 frequent SNP copy number change and LOH involving 1, 2p, 3, 4q, 5q, 6, 7p, 8, 9, 10, 11, 12q, 13, 14, 16q, 17, and 20, (2 reduced SNP copy number at FAT gene (4q35 in 75% of SS cases, and (3 the separation of all SS cases from normal control samples by SNP LOH gene clusters at chromosome regions of 9q31q34, 10p11q26, and 13q11q12. These findings provide some intriguing information for our current understanding of the molecular pathogenesis of this tumour and suggest the possibility of presence of functional SNP LOH in SS tumour cells.

  6. Acyl-CoA synthetase long-chain 5 genotype is associated with body composition changes in response to lifestyle interventions in postmenopausal women with overweight and obesity: a genetic association study on cohorts Montréal-Ottawa New Emerging Team, and Complications Associated with Obesity.

    Science.gov (United States)

    Rajkumar, Abishankari; Lamothe, Gilles; Bolongo, Pierrette; Harper, Mary-Ellen; Adamo, Kristi; Doucet, Éric; Rabasa-Lhoret, Remi; Prud'homme, Denis; Tesson, Frédérique

    2016-08-11

    Genetic studies on Acyl-CoA Synthetase Long-Chain 5 (ACSL5) demonstrate an association between rs2419621 genotype and rate of weight loss in women with obesity in response to caloric restriction. Our objectives were to (1) confirm results in two different populations of women with overweight and obesity (2) study rs2419621's influence on body composition parameters of women with overweight and obesity following lifestyle interventions. rs2419621 genotype was determined in women with overweight and obesity who participated in the Montréal-Ottawa New Emerging Team (MONET n = 137) and Complications Associated with Obesity (CAO n = 37) studies. Genotyping was done using TaqMan MGB probe-based assay. Multiple linear regression analyses were used to test for associations. When studying women with overweight and obesity, rs2419621 [T] allele carriers had a significantly greater decrease in visceral fat, absolute and percent fat mass and a greater increase in percent lean mass in response to lifestyle intervention in comparison to non-carriers. Studying only individuals with obesity showed similar results with rs2419621 [T] allele carriers also displaying a significantly greater decrease in body mass index following the lifestyle intervention in comparison to non-carriers. Women with overweight and obesity carrying the ACSL5 rs2419621 [T] allele are more responsive to lifestyle interventions in comparison to non-carriers. Conducting such genetic association studies can aid in individualized treatments/interventions catered towards an individual's genotype.

  7. Elevated Levels of Endocannabinoids in Chronic Hepatitis C May Modulate Cellular Immune Response and Hepatic Stellate Cell Activation

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    Eleonora Patsenker

    2015-03-01

    Full Text Available The endocannabinoid (EC system is implicated in many chronic liver diseases, including hepatitis C viral (HCV infection. Cannabis consumption is associated with fibrosis progression in patients with chronic hepatitis C (CHC, however, the role of ECs in the development of CHC has never been explored. To study this question, anandamide (AEA and 2-arachidonoyl glycerol (2-AG were quantified in samples of HCV patients and healthy controls by gas and liquid chromatography mass spectrometry. Fatty acid amide hydrolase (FAAH and monoaclyglycerol lipase (MAGL activity was assessed by [3H]AEA and [3H]2-AG hydrolysis, respectively. Gene expression and cytokine release were assayed by TaqMan PCR and ELISpot, respectively. AEA and 2-AG levels were increased in plasma of HCV patients, but not in liver tissues. Hepatic FAAH and MAGL activity was not changed. In peripheral blood mononuclear cells (PBMC, ECs inhibited IFN-γ, TNF-α, and IL-2 secretion. Inhibition of IL-2 by endogenous AEA was stronger in PBMC from HCV patients. In hepatocytes, 2-AG induced the expression of IL-6, -17A, -32 and COX-2, and enhanced activation of hepatic stellate cells (HSC co-cultivated with PBMC from subjects with CHC. In conclusion, ECs are increased in plasma of patients with CHC and might reveal immunosuppressive and profibrogenic effects.

  8. Validation of a Real Time PCR for Classical Swine Fever Diagnosis

    Science.gov (United States)

    Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Érica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves

    2014-01-01

    The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5′NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5′NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF. PMID:24818039

  9. Association analysis of calpain 10 gene variants/haplotypes with gestational diabetes mellitus among Mexican women.

    Science.gov (United States)

    Castro-Martínez, Anna Gabriela; Sánchez-Corona, José; Vázquez-Vargas, Adriana Patricia; García-Zapién, Alejandra Guadalupe; López-Quintero, Andres; Villalpando-Velazco, Héctor Javier; Flores-Martínez, Silvia Esperanza

    2018-02-28

    Gestational diabetes mellitus (GDM) is a metabolically complex disease with major genetic determinants. GDM has been associated with insulin resistance and dysfunction of pancreatic beta cells, so the GDM candidate genes are those that encode proteins modulating the function and secretion of insulin, such as that for calpain 10 (CAPN10). This study aimed to assess whether single nucleotide polymorphism (SNP)-43, SNP-44, SNP-63, and the indel-19 variant, and specific haplotypes of the CAPN10 gene were associated with gestational diabetes mellitus. We studied 116 patients with gestational diabetes mellitus and 83 women with normal glucose tolerance. Measurements of anthropometric and biochemical parameters were performed. SNP-43, SNP-44, and SNP-63 were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphisms, while the indel-19 variant was detected by TaqMan qPCR assays.  The allele, genotype, and haplotype frequencies of the four variants did not differ significantly between women with gestational diabetes mellitus and controls. However, in women with gestational diabetes mellitus, glucose levels were significantly higher bearing the 3R/3R genotype than in carriers of the 3R/2R genotype of the indel-19 variant (p = 0.006). In conclusion, the 3R/3R genotype of the indel-19 variant of the CAPN-10 gene influenced increased glucose levels in these Mexican women with gestational diabetes mellitus.

  10. Development and validation of a real-time PCR for Chlamydia suis diagnosis in swine and humans.

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    Kristien De Puysseleyr

    Full Text Available Pigs are the natural host for Chlamydia suis, a pathogen which is phylogenetically highly related to the human pathogen C. trachomatis. Chlamydia suis infections are generally treated with tetracyclines. In 1998, tetracyline resistant C. suis strains emerged on U.S. pig farms and they are currently present in the Belgian, Cypriote, German, Israeli, Italian and Swiss pig industry. Infections with tetracycline resistant C. suis strains are mainly associated with severe reproductive failure leading to marked economical loss. We developed a sensitive and specific TaqMan probe-based C. suis real-time PCR for examining clinical samples of both pigs and humans. The analytical sensitivity of the real-time PCR is 10 rDNA copies/reaction without cross-amplifying DNA of other Chlamydia species. The PCR was successfully validated using conjunctival, pharyngeal and stool samples of slaughterhouse employees, as well as porcine samples from two farms with evidence of reproductive failure and one farm without clinical disease. Chlamydia suis was only detected in diseased pigs and in the eyes of humans. Positive humans had no clinical complaints. PCR results were confirmed by culture in McCoy cells. In addition, Chlamydia suis isolates were also examined by the tet(C PCR, designed for demonstrating the tetracycline resistance gene tet(C. The tet(C gene was only present in porcine C. suis isolates.

  11. A common variant of MAF/c-MAF, transcriptional factor gene in the kidney, is associated with gout susceptibility.

    Science.gov (United States)

    Higashino, Toshihide; Matsuo, Hirotaka; Okada, Yukinori; Nakashima, Hiroshi; Shimizu, Seiko; Sakiyama, Masayuki; Tadokoro, Shin; Nakayama, Akiyoshi; Kawaguchi, Makoto; Komatsu, Mako; Hishida, Asahi; Nakatochi, Masahiro; Ooyama, Hiroshi; Imaki, Junko; Shinomiya, Nariyoshi

    2018-01-01

    Gout is a multifactorial disease characterized by acute inflammatory arthritis, and it is caused as a consequence of hyperuricemia. A recent meta-analysis of genome-wide association studies has newly identified the relationship between serum uric acid (SUA) levels and rs889472, a single nucleotide polymorphism of musculoaponeurotic fibrosarcoma oncogene (MAF/c-MAF). However, it remained unclear whether rs889472 is associated with gout susceptibility. In the present study, we investigate the association between c-MAF rs889472 and gout in Japanese male population. We genotyped 625 male patients who were clinically diagnosed as gout and 1221 male control subjects without hyperuricemia or a history of gout by TaqMan method. As a result, the major allele (C), which reportedly increases SUA levels, had a higher frequency in the gout cases (58.8%) than in the controls (55.0%). A logistic regression analysis showed a significant association between rs889472 and gout (p = 0.029, odds ratio = 1.17; 95% confidence interval 1.02-1.34). C-MAF is reported as a pivotal transcriptional factor in the development and differentiation of renal proximal tubular cells. Because urate is mainly regulated in renal proximal tubular cells, c-MAF may have an important role in urate regulation in the kidney and influence not only SUA but also gout susceptibility. Our finding shows that rs889472 of c-MAF is associated with gout susceptibility.

  12. Associations between interleukin and interleukin receptor gene polymorphisms and risk of gout.

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    Liu, Shiguo; Zhou, Zheng; Wang, Can; Guo, Mingzhen; Chu, Nan; Li, Changgui

    2015-09-24

    Gout is a self-limiting, auto-inflammatory arthritis induced by the deposition of monosodium urate crystals in the synovial fluid and periarticular tissues. The aim of this study was to investigate the associations between genetic variants in the interleukin (IL) and interleukin receptor (ILR) genes IL-33, IL-1RL1, IL-23R, and signal transducer and activator of transcription 4 (STAT4) and susceptibility to gout in Chinese Han male individuals. The genetic distributions of rs3939286 in IL-33, rs13015714 in IL-1RL1, rs10889677 in IL-23R, and rs7574865 in STAT4 were detected in 1100 men with gout and 1227 ethnically matched controls, using Taqman allelic discrimination real-time polymerase chain reaction (PCR). Differences in these polymorphisms between the groups were investigated using χ(2) tests. The genotype-phenotype relationship among gout patients was tested by analysis of variance. There was a significant difference in genotypic frequencies of IL-23R rs10889677 between gout patients and controls (χ(2) = 81.386, P gout in Chinese Han male individuals. However, further studies in other ethnic groups are needed to confirm these results.

  13. The rs7517847 polymorphism in the IL-23R gene is associated with gout in a Chinese Han male population.

    Science.gov (United States)

    Liu, Shiguo; He, Hongmei; Yu, Renchao; Han, Lin; Wang, Can; Cui, Ying; Li, Changgui

    2015-05-01

    Gout is a polygenic auto-inflammatory disease, in which inflammation plays an important role in disease pathogenesis. The cytokine interleukin (IL)-23 promotes inflammation and helps to guide inflammatory cells, while studies have shown that the IL-23R gene is associated with susceptibility to several immune-related diseases. This study aimed to determine whether the IL-23R rs7517847 (G/T) polymorphism is associated with gout in a Chinese Han male population. We recruited 400 patients with gout and 582 gout-free controls. After obtaining blood samples for DNA extraction, genotyping of the rs7517847 polymorphism was performed by fluorescence-based quantitative PCR using TaqMan probes. An association analysis was carried out using the χ(2) test. A genotype-phenotype analysis was also conducted. Both genotypic and allelic frequencies of rs7517847 differed significantly between gout patients and controls (χ(2) = 6.792, df = 2, P = 0.034 by genotype; χ(2) = 4.202, df = 1, P = 0.04 by allele). IL-23R may be associated with gout in a Chinese Han male population, although our findings should be confirmed using larger sample sizes and other independent populations.

  14. Identification of rs671, a common variant of ALDH2, as a gout susceptibility locus.

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    Sakiyama, Masayuki; Matsuo, Hirotaka; Nakaoka, Hirofumi; Yamamoto, Ken; Nakayama, Akiyoshi; Nakamura, Takahiro; Kawai, Sayo; Okada, Rieko; Ooyama, Hiroshi; Shimizu, Toru; Shinomiya, Nariyoshi

    2016-05-16

    Gout is a common disease resulting from hyperuricemia. Recently, a genome-wide association study identified an association between gout and a single nucleotide polymorphism (SNP) rs2188380, located on an intergenic region between MYL2 and CUX2 on chromosome 12. However, other genes around rs2188380 could possibly be gout susceptibility genes. Therefore, we performed a fine-mapping study of the MYL2-CUX2 region. From 8,595 SNPs in the MYL2-CUX2 region, 9 tag SNPs were selected, and genotyping of 1,048 male gout patients and 1,334 male controls was performed by TaqMan method. Eight SNPs showed significant associations with gout after Bonferroni correction. rs671 (Glu504Lys) of ALDH2 had the most significant association with gout (P = 1.7 × 10(-18), odds ratio = 0.53). After adjustment for rs671, the other 8 SNPs no longer showed a significant association with gout, while the significant association of rs671 remained. rs671 has been reportedly associated with alcohol drinking behavior, and it is well-known that alcohol drinking elevates serum uric acid levels. These data suggest that rs671, a common functional SNP of ALDH2, is a genuine gout-associated SNP in the MYL2-CUX2 locus and that "A" allele (Lys) of rs671 plays a protective role in the development of gout.

  15. Development and validation of a real-time PCR assay for specific and sensitive detection of canid herpesvirus 1.

    Science.gov (United States)

    Decaro, Nicola; Amorisco, Francesca; Desario, Costantina; Lorusso, Eleonora; Camero, Michele; Bellacicco, Anna Lucia; Sciarretta, Rossana; Lucente, Maria Stella; Martella, Vito; Buonavoglia, Canio

    2010-10-01

    A TaqMan-based real-time PCR assay targeting the glycoprotein B-encoding gene was developed for diagnosis of canid herpesvirus 1 (CHV-1) infection. The established assay was highly specific, since no cross-reactions were observed with other canine DNA viruses, including canine parvovirus type 2, canine minute virus, or canine adenovirus types 1 and 2. The detection limit was 10(1) and 1.20 x 10(1) DNA copies per 10 microl(-1) of template for standard DNA and a CHV-1-positive kidney sample, respectively: about 1-log higher than a gel-based PCR assay targeting the thymidine kinase gene. The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. CHV-1 isolates of different geographical origins were recognised by the TaqMan assay. Tissues and clinical samples collected from three pups which died of CHV-1 neonatal infection were also tested, displaying a wide distribution of CHV-l DNA in their organs. Unlike other CHV-1-specific diagnostic methods, this quantitative assay permits simultaneous detection and quantitation of CHV-1 DNA in a wide range of canine tissues and body fluids, thus providing a useful tool for confirmation of a clinical diagnosis, for the study of viral pathogenesis and for evaluation of the efficacy of vaccines and antiviral drugs. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  16. Performance and Logistical Challenges of Alternative HIV-1 Virological Monitoring Options in a Clinical Setting of Harare, Zimbabwe

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    Pascale Ondoa

    2014-01-01

    Full Text Available We evaluated a low-cost virological failure assay (VFA on plasma and dried blood spot (DBS specimens from HIV-1 infected patients attending an HIV clinic in Harare. The results were compared to the performance of the ultrasensitive heat-denatured p24 assay (p24. The COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, served as the gold standard. Using a cutoff of 5,000 copies/mL, the plasma VFA had a sensitivity of 94.5% and specificity of 92.7% and was largely superior to the VFA on DBS (sensitivity = 61.9%; specificity = 99.0% or to the p24 (sensitivity = 54.3%; specificity = 82.3% when tested on 302 HIV treated and untreated patients. However, among the 202 long-term ART-exposed patients, the sensitivity of the VFA decreased to 72.7% and to 35.7% using a threshold of 5,000 and 1,000 RNA copies/mL, respectively. We show that the VFA (either on plasma or on DBS and the p24 are not reliable to monitor long-term treated, HIV-1 infected patients. Moreover, achieving acceptable assay sensitivity using DBS proved technically difficult in a less-experienced laboratory. Importantly, the high level of virological suppression (93% indicated that quality care focused on treatment adherence limits virological failure even when PCR-based viral load monitoring is not available.

  17. The role of polymorphisms associated with early tooth eruption in dental and occlusal traits in East Asian populations

    Science.gov (United States)

    Kawaguchi, Akira; Kim, Yong-Il; Haga, Shugo; Katayama, Koshu; Ishida, Hajime; Park, Soo-Byung; Maki, Koutaro; Kimura, Ryosuke

    2014-01-01

    Objective A recent study suggested that rs6504340, a polymorphism within the homeobox B (HOXB) gene cluster, is associated with the susceptibility for malocclusions in Europeans. The resulting malocclusions require orthodontic treatment. The aim of this study was to investigate the association of rs6504340 and other dentition-implicated polymorphisms with dental and occlusal traits in Korean and Japanese populations. Methods The study participants included 223 unrelated Koreans from the Busan area and 256 unrelated Japanese individuals from the Tokyo metropolitan area. DNA samples were extracted from saliva specimens. Genotyping for rs6504340 and four single nucleotide polymorphisms (SNPs) that have been shown to be associated with the timing of first tooth eruption and the number of teeth at 1 year of age (rs10506525, rs1956529, rs9674544, and rs8079702) was performed using TaqMan assays. The Index of Orthodontic Treatment Need (IOTN), overjet, overbite, arch length discrepancy, crown sizes, and length and width of the dental arches were measured. Spearman's correlation coefficients were calculated to evaluate relationships between rs6504340 and these dental/occlusal traits. Results We evaluated the aesthetic components and dental health components of the IOTN in the Korean and Japanese populations and found that neither rs6504340 nor the other four SNPs showed any association with dental and occlusal traits in these East Asian populations. Conclusions These negative results suggest that further research is needed to identify the genetic determinants of malocclusions in order to reach a consensus. PMID:24696826

  18. Impact of two different commercial DNA extraction methods on BK virus viral load

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    Massimiliano Bergallo

    2016-03-01

    Full Text Available Background and aim: BK virus, a member of human polyomavirus family, is a worldwide distributed virus characterized by a seroprevalence rate of 70-90% in adult population. Monitoring of viral replication is made by evaluation of BK DNA by quantitative polymerase chain reaction. Many different methods can be applied for extraction of nucleic acid from several specimens. The aim of this study was to assess the impact of two different DNA extraction procedure on BK viral load. Materials and methods: DNA extraction procedure including the Nuclisens easyMAG platform (bioMerieux, Marcy l’Etoile, France and manual QIAGEN extraction (QIAGEN Hilden, Germany. BK DNA quantification was performed by Real Time TaqMan PCR using a commercial kit. Result and discussion: The samples capacity, cost and time spent were compared for both systems. In conclusion our results demonstrate that automated nucleic acid extraction method using Nuclisense easyMAG was superior to manual protocol (QIAGEN Blood Mini kit, for the extraction of BK virus from serum and urine specimens.

  19. Exploiting 16S rRNA gene for the detection and quantification of fish as a potential allergenic food: A comparison of two real-time PCR approaches.

    Science.gov (United States)

    Fernandes, Telmo J R; Costa, Joana; Oliveira, M Beatriz P P; Mafra, Isabel

    2018-04-15

    Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. In this work, two real-time PCR systems based on the EvaGreen dye and a TaqMan probe are proposed and compared. New primers were designed to target the 16S rRNA gene, as a universal maker for fish detection, with fully demonstrated specificity for a wide range of fish species. Both systems showed similar absolute sensitivities, down to 0.01 pg of fish DNA, and adequate real-time PCR performance parameters. The probe system showed higher relative sensitivity and dynamic range (0.0001-50%) than the EvaGreen (0.05-50%). They were both precise, but trueness was compromised at the highest tested level with the EvaGreen assay. Therefore, both systems were successful, although the probe one exhibited the best performance. Its application to verify labelling compliance of foodstuffs suggested a high level of mislabelling and/or fraudulent practices. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Functional promoter rs2868371 variant of HSPB1 associates with radiation-induced esophageal toxicity in patients with non-small-cell lung cancer treated with radio(chemo)therapy

    International Nuclear Information System (INIS)

    Lopez Guerra, Jose Luis; Wei Qingyi; Yuan Xianglin; Gomez, Daniel; Liu Zhensheng; Zhuang Yan; Yin Ming; Li Minghuan; Wang, Li-E; Cox, James D.; Liao Zhongxing

    2011-01-01

    Purpose: We investigated the association between single-nucleotide polymorphisms (SNPs) in the heat shock protein beta-1 (HSPB1) gene and the risk of radiation-induced esophageal toxicity (RIET) in patients with non-small-cell lung cancer (NSCLC). Materials and methods: The experimental dataset comprised 120 NSCLC patients who were treated with radio(chemo)therapy between 2005 and 2009, when novel radiation techniques were implemented at MD Anderson. The validation dataset comprised 181 NSCLC patients treated between 1998 and 2004. We genotyped two SNPs of the HSPB1 gene (rs2868370 and rs2868371) by TaqMan assay. Results: Univariate and multivariate analyses of the experimental dataset showed that the CG/GG genotypes of HSPB1 rs2868371 were associated with significantly lower risk of grade ⩾3 RIET than the CC genotype (univariate hazard ratio [HR] 0.30; 95% confidence interval [CI], 0.10–0.91; P = 0.033; multivariate HR 0.29; 95% CI, 0.09–0.97; P = 0.045). This difference in risk was replicated in the validation cohort despite the different radiation techniques used during that period. Conclusions: The CG/GG genotypes of HSPB1 rs2868371 were associated with lower risk of RIET, compared with the CC genotype in patients with NSCLC treated with radio(chemo)therapy. This finding should be validated in large multi-institutional prospective trials.