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PCR Inhibitors (pcr + inhibitor)
Selected AbstractsMulticenter quality control of the detection of HIV-1 genome in semen before medically assisted procreationJOURNAL OF MEDICAL VIROLOGY, Issue 7 2006Christophe Pasquier Abstract Couples in whom the man is HIV-1-positive may use medically assisted procreation in order to conceive a child without contaminating the female partner. But, before medically assisted procreation, the semen has to be processed to exclude HIV and tested for HIV nucleic acid before and after processing. The performance was evaluated of the technical protocols used to detect and quantify HIV-1 in 11 centers providing medically assisted procreation for couples with HIV-1 infected men by testing panels of seminal plasma and cells containing HIV-1 RNA and/or DNA. The performance of these tests varied due to the different assays used. False positive results were obtained in 14,19% of cases. The sensitivity for RNA detection in seminal plasma was 500,1,000 RNA copies/ml, over 500 RNA copies/106 cells in semen cells, and for DNA detection in semen cells 50,500 DNA copies/106 cells. The use of silica-based extraction seemed to increase the assay performance, whereas the use of internal controls to detect PCR inhibitor did not. This first quality control highlights the need for technical improvements of the assays to detect and quantify HIV in semen fractions and for regular evaluation of their performance. J. Med. Virol. 78:877,882, 2006. © 2006 Wiley-Liss, Inc. [source] Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environmentsENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Juan M. Gonzalez Summary Microbial assessment of natural biodiversity is usually achieved through polymerase chain reaction (PCR) amplification. Deoxyribonucleic acid (DNA) sequences from natural samples are often difficult to amplify because of the presence of PCR inhibitors or to the low number of copies of specific sequences. In this study, we propose a non-specific preamplification procedure to overcome the presence of inhibitors and to increase the number of copies prior to carrying out standard amplification by PCR. The pre-PCR step is carried out through a multiple displacement amplification (MDA) technique using random hexamers as priming oligonucleotides and ,29 DNA polymerase in an isothermal, whole-genome amplification reaction. Polymerase chain reaction amplification using specific priming oligonucleotides allows the selection of the sequences of interest after a preamplification reaction from complex environmental samples. The procedure (MDA-PCR) has been tested on a natural microbial community from a hypogean environment and laboratory assemblages of known bacterial species, in both cases targeting the small subunit ribosomal RNA gene sequences. Results from the natural community showed successful amplifications using the two steps protocol proposed in this study while standard, direct PCR amplification resulted in no amplification product. Amplifications from a laboratory assemblage by the two-step proposed protocol were successful at bacterial concentrations ,,10-fold lower than standard PCR. Amplifications carried out in the presence of different concentrations of fulvic acids (a soil humic fraction) by the MDA-PCR protocol generated PCR products at concentrations of fulvic acids over 10-fold higher than standard PCR amplifications. The proposed procedure (MDA-PCR) opens the possibility of detecting sequences represented at very low copy numbers, to work with minute samples, as well as to reduce the negative effects on PCR amplifications of some inhibitory substances commonly found in environmental samples. [source] Development of real-time detection direct test for hepatitis B virus and comparison with two commercial tests using the WHO international standardJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 11 2003MOTOKAZU MUKAIDE Abstract Aims:, A highly reproducible and sensitive hepatitis B virus real-time detection direct (HBV RTD-direct) test using DNA extraction by magnetic beads coated with polyclonal anti-HBsAg, followed by the real-time detection polymerase chain reaction (PCR) method, was developed for the detection of HBV DNA. Methods:, The HBV DNA could be extracted from the HBsAg positive viral particles without resulting in viral DNA fragmentation. The HBV RTD-direct test was validated using a serial dilution panel of the WHO standard HBV DNA 97/746 I. Results:, The test had a dynamic range of 0.7,8.0 log10 international units (IU) per mL and the results were shown to be comparable to those obtained with two commercially available tests: the HBV DNA transcription-mediated amplification-hybridization protection assay and the Amplicor HBV Monitor test. In addition, the HBV RTD-direct test, based on magnetic extraction, successfully eliminated PCR inhibitors in clinical specimens. Conclusion:, We conclude that the HBV RTD-direct test is an excellent alternative for monitoring patients undergoing antiviral treatment or for screening various clinical specimens. [source] Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre-emptive therapyJOURNAL OF MEDICAL VIROLOGY, Issue 1 2009Céline Bressollette-Bodin Abstract Two quantitative duplex real-time PCR assays were developed for co-amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 106 peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5,×,109 PBLs/L. Albumin co-amplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre-emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article. J. Med. Virol. 81:90,98, 2009. © 2008 Wiley-Liss, Inc. [source] Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheeseLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2007P. Cremonesi Abstract Aim:, To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. Methods and Results:, A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g,1 for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. Conclusions:,Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. Significance and Impact of the Study:, This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure. [source] A combined agar-absorption and BIO-PCR assay for rapid, sensitive detection of Xylella fastidiosa in grape and citrusPLANT PATHOLOGY, Issue 1 2005M. Fatmi Application of the polymerase chain reaction (PCR) to disease diagnosis is limited in part by the presence of PCR inhibitors. Inhibition can be overcome and sensitivity increased by culturing bacteria on agar media prior to PCR (termed BIO-PCR). However, Xylella fastidiosa grows slowly, requiring 10,14 days for visible colonies to appear. In this study an agar-absorption BIO-PCR method for detecting X. fastidiosa in grape and citrus plants was developed. Optimum lengths of time for absorption of inhibitors by the agar medium or enrichment of bacteria on the medium were determined for Pierce's disease of grape and citrus variegated chlorosis. When petioles of grape and citrus leaves with symptoms were spotted onto agar media, the spots washed after various time intervals and assayed for X. fastidiosa by real-time PCR, 97% (31 out of 32) and 100% (six out of six) of spots were positive after 2 days and 4 h for grape and citrus, respectively. With direct PCR, only 12·5% (four out of 32) and 33% (two out of six) of spots were positive, respectively, and visible X. fastidiosa colonies were evident after 10 and 14 days, respectively. In a separate experiment with samples from a different vineyard, 46% (13 out of 28) of the grape samples (agar spots) were positive after 1 day and 93% (26 out of 28) after 5 days using agar-absorption PCR. In contrast, all samples were negative by direct PCR. Viable X. fastidiosa were recovered from all samples after 14 days. Further tests with eight randomly selected grape petioles from three Texas vineyards known to have Pierce's disease resulted in 50% being positive by a simple 24 h agar-absorption PCR assay, whereas none was positive by direct PCR. Overall, 10 out of 16 (63%) vines from five vineyards (two in California and three in Texas) were positive after the 24 h agar-absorption PCR assay. In contrast, only one vine was positive by direct PCR. This simple agar absorption-based PCR assay protocol should prove useful for the routine detection of X. fastidiosa and other slow-growing bacteria in the presence of PCR inhibitors. [source] Technical note: PCR analysis of minimum target amount of ancient DNAAMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY, Issue 2 2010Daniela Woide Abstract The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low-volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and ,-actin gene specific fragments were amplified via low-volume PCR in a total reaction volume of 1 ,l. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low-volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley-Liss, Inc. [source] A highly sensitive and quantitative telomerase activity assay with pancreatic juice is useful for diagnosis of pancreatic carcinoma without problems due to polymerase chain reaction inhibitorsCANCER, Issue 10 2004Analysis of 100 samples of pancreatic juice from consecutive patients Abstract BACKGROUND Early detection of pancreatic carcinoma is difficult even with current diagnostic tools. Novel biomarkers and detection techniques are urgently needed. Telomerase activity is a promising diagnostic marker. However, the conventional telomeric repeat amplification protocol (TRAP) assay is not suitable for clinical application because of its complexity, time-consuming nature, and the effects of polymerase chain reaction (PCR) inhibitors in samples leading to difficulties in quantification. METHODS The authors used a hybridization protection assay in combination with TRAP (TRAP/HPA) to investigate the effects of PCR inhibitors in pancreatic juice on quantification of telomerase activity. They analyzed 117 consecutive samples of pancreatic juice to determine the feasibility of TRAP/HPA for diagnosis of pancreatic carcinoma. RESULTS The authors found that TRAP/HPA was 1000-fold more sensitive than the conventional TRAP assay, and that the effects of PCR inhibitors could be avoided by diluting samples. In a large analysis of pancreatic juice samples with TRAP/HPA, 17 samples were excluded from the final analysis because of insufficient follow-up periods or inadequate treatment of the samples. Relative telomerase activity (RTA) in samples from patients with pancreatic carcinoma was significantly higher in comparison to samples from patients with pancreatitis and 13 (61.9%) of 21 samples from patients with pancreatic carcinoma showed high RTA (> 4 U). Meanwhile, high RTAs were observed in 4 of 35 (11.4%) samples from patients with intraductal papillary mucinous tumor and in 1 of 40 samples (2.5%) fom patients without malignant disease. CONCLUSIONS TRAP/HPA accurately evaluated weak telomerase activity in pancreatic juice samples without the problem due to PCR inhibitors. This large analysis of nonselected pancreatic juice samples suggested that TRAP/HPA is a promising approach for the diagnosis of pancreatic carcinoma. Cancer 2004. © 2004 American Cancer Society. [source] | |||||||||||||