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TaqMan PCR (taqman + pcr)
Selected AbstractsQuantitation of cytomegalovirus (CMV) DNA by real-time PCR for occurrence of CMV disease in HIV-infected patients receiving highly active antiretroviral therapyJOURNAL OF MEDICAL VIROLOGY, Issue 3 2003Karine Gourlain Abstract In HIV-infected patients treated with highly active antiretroviral therapy (HAART) included in the Predivir cohort, we have evaluated the usefulness of CMV DNA quantitation by a TaqMan® PCR assay from peripheral blood leukocytes (PBLs) to predict CMV disease occurrence. In parallel with the immune restoration after treatment by HAART, the percentage of positive samples decreased progressively from 7.3% at Day 0 to 3.5% at Month 12. Among the CMV markers, the smallest concordance with PBL CMV TaqMan® PCR, as evaluated by kappa, was observed with pp65 antigenemia, whereas concordance with all other CMV markers was high. Among the 16 patients with CMV DNA copies at least once >100/150,000 cells, CMV disease occurred in six during follow-up, whereas among the 159 patients with CMV DNA copies always <10/150,000 cells, CMV disease occurred in three and among the seven patients with CMV DNA copies >10 and <100 occurred in only one. In univariate Cox models, all the CMV markers including PBL CMV TaqMan® PCR >10/150,000 cells (RR: 27.6, IC95: 7.1,107.2), the CD4 cell count <75 cells/mm3 and the HIV viral load >100,000 copies/ml were predictive for CMV disease. In a stepwise multivariate analysis, which should be interpreted with caution due to the small number of events (n = 10), three covariates were associated independently with CMV disease: pp65 antigenemia >100 nuclei/200,000, PBL CMV TaqMan® PCR >10 copies/150,000 cells and HIV viral load remaining or increasing >100,000 copies/ml. J. Med. Virol. 69:401,407, 2003. © 2003 Wiley-Liss, Inc. [source] Parvovirus B19 infection in pregnancy: Quantitative viral DNA analysis using a kinetic fluorescence detection system (TaqMan PCR)JOURNAL OF MEDICAL VIROLOGY, Issue 2 2002Antje Knöll M.D. Abstract Human parvovirus B19 infections are common in the general population, and infection during pregnancy may cause hydrops fetalis and fetal death. To initiate adequate treatment, accurate laboratory diagnosis is essential. The most sensitive tests are nested PCR systems, but these assays provide semiquantitative results at best. A parvovirus B19 DNA assay was developed based on the real time TaqMan PCR. This method was calibrated on the basis of serial plasmid dilutions and tested with an international parvovirus B19 standard. The assay was capable of quantifying parvovirus B19 DNA from one to about 5,×,107 genome equivalents per reaction (corresponding to 100 to 5,×,109 genome equivalents per ml serum). Samples from 51 pregnant women with suspected acute parvovirus B19 infection were tested, and positive PCR results were obtained in at least one of the materials investigated in 41 cases. The median viral DNA load in maternal blood samples was 1.3,×,104 copies/ml (range 7.2,×,102,2.6,×,107). Maternal virus DNA concentration was not associated with the presence of maternal symptoms and/or fetal complications. As the stage of infection was not known in the majority of cases, our data do not exclude an association between peak levels of parvovirus B19 DNA and the development of complications. Maternal sera and corresponding fetal material were available for concurrent testing from 15 DNA-positive cases: in most fetal samples, viral DNA concentrations were several orders of magnitude higher (up to 2.1,×,1012 copies/ml) compared to the corresponding maternal blood samples. J. Med. Virol. 67:259,266, 2002. © 2002 Wiley-Liss, Inc. [source] Langerhans cell histiocytosis and human herpes virus 6 (HHV-6), an analysis by real-time polymerase chain reactionJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2006Michael P. Glotzbecker Abstract Patients with Langerhans cell histiocytosis (LCH) usually present to orthopedic surgeons because this disease most commonly affects bone. The pathogenesis of LCH is unknown, although roles for environmental, infectious, immunologic, and genetic causes have been postulated. More specifically, there is limited data suggesting that human herpes virus 6 (HHV-6) may be a potential etiologic agent. Frozen biopsy material was obtained from 13 patients with LCH and 20 patients without the disease. After ensuring histologic adequacy of the material, the tissue was tested for HHV-6 by qualitative and quantitative real-time TaqMan PCR. Four of 13 patients with LCH had evidence of HHV-6 DNA in their tissue while 7 of 20 control patients tested positive for HHV-6 genome. Viral loads are reported for the positive patients; no statistical difference was observed in the presence or quantity of HHV-6 DNA found in either population, suggesting that the prevalence of HHV-6 in the tissue of LCH patients is the same as that found in tissue from individuals without disease. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:313,320, 2006 [source] Detection of Phytophthora nicotianae in Soil with Real-time Quantitative PCRJOURNAL OF PHYTOPATHOLOGY, Issue 1 2010Junli Huang Abstract Phytophthora nicotianae is one of the most important soil-borne plant pathogens. A rapid, specific and sensitive real-time polymerase chain reaction (PCR) detection method for P. nicotianae was established, which used primers targeting the internal transcribed spacer (ITS) regions of rDNA genes of Phytophthora spp. Based on the nucleotide sequences of ITS2 of 15 different species of Phytophthora, the primers and probe were designed specifically to amplify DNA from P. nicotianae. With a series of 10-fold DNA dilutions extracted from P. nicotianae pure cultures, the detection limit was 10 pg/,l in conventional PCR, whereas in SYBR Green I PCR the detection limit was 0.12 fg/,l and in TaqMan PCR 1.2 fg/,l, and real-time PCR was 104,105 times more sensitive than conventional PCR. The simple and rapid procedures maximized the yield and quality of recovered DNA from soil and allowed the processing of many samples in a short time. The direct DNA extractions from soil were utilized to yield DNA suitable for PCR. By combining this protocol with the real-time PCR procedure it has been possible to specifically detect P. nicotianae in soil, and the degree of sensitivity was 1.0 pg/,l. The system was applied to survey soil samples from tobacco field sites in China for the presence of P. nicotianae and the analyses of naturally infested soil showed the reliability of the real-time PCR method. [source] ORIGINAL ARTICLE: No Association Between the GSTP1 Exon 5 Polymorphism and Susceptibility to Advanced Stage Endometriosis in the Korean PopulationAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 3 2010Myung Jae Jeon CitationJeon MJ, Choi YM, Hong MA, Lee GH, Ku SY, Kim SH, Kim JG, Moon SY. No Association between the GSTP1 exon 5 polymorphism and susceptibility to advanced stage endometriosis in the Korean population. Am J Reprod Immunol 2010; 63: 222,226 Problem, To investigate whether the glutathione- S -transferase P1 (GSTP1) exon 5 polymorphism is associated with susceptibility to advanced stage endometriosis in Korean women. Method of study, Case,control study in a collective of 260 patients and 164 controls. Genotyping of the GSTP1 exon 5 polymorphism was performed by using real-time TaqMan PCR assay. Results, The genotype distribution of the GSTP1 exon 5 polymorphism in the endometriosis group was not significantly different from that of the control group (AA/AG/GG rates were 64.2%/32.7%/3.1% and 65.2%/31.7%/3.0% for the endometriosis and control groups, respectively, P = 0.977). Further subgroup analysis according to either stage or bilaterality of ovarian endometrioma also found no significant difference in the genotype distribution between any of the endometriosis subgroups and the control group. Conclusion, These findings suggest that the GSTP1 exon 5 polymorphism is not a major determinant of the development of advanced stage endometriosis in the Korean population. [source] The novel use of the human nasal epithelial cell line RPMI 2650 as an in vitro model to study the influence of allergens and cytokines on transforming growth factor-, gene expression and protein releaseCLINICAL & EXPERIMENTAL ALLERGY, Issue 6 2005R. J. Salib Summary Background The epithelial accumulation of mast cells is a feature of allergic rhinitis and this has been linked to the expression of the known mast cell chemoattractant transforming growth factor-, (TGF-,) at this site. Little is known concerning the regulation of TGF-, gene expression or protein release by nasal epithelial cells. To address this we have utilized the RPMI 2650 human nasal epithelial cell line, which has some features that closely resemble normal nasal epithelium and has been reported to secrete a TGF-,-like molecule. Objectives To investigate the regulation of TGF-, gene expression and protein secretion in RPMI 2650 nasal epithelial cells following exposure to allergens (house dust mite (HDM) and grass pollen) and mast cell associated T-helper type 2 (Th2) cytokines (IL-4, IL-13, and TNF-,). Methods Light and scanning electron microscopy was used to evaluate the morphology of RPMI 2650 cells in culture, enzyme-linked immunosorbent assay was used to investigate their TGF-, secretory capacity and the identification of the TGF-, isotype(s) involved, flow cytometry was used to demonstrate the presence of TGF-, receptors on the RPMI 2650 cells, and the quantitative real-time TaqMan PCR was used to measure TGF-, gene expression. Results TGF-,2 was identified as the main isotype secreted by the RPMI 2650 cells. HDM allergens and TNF-, increased both TGF-, gene expression and protein release from these cells, whereas grass pollen, IL-4, and IL-13 were without effect. Conclusions The RPMI 2650 nasal epithelial cell line represents a valid in vitro model to evaluate the regulation of TGF-, biology. In this system HDM allergens have stimulatory activity that is fundamentally different from that of grass pollen allergens, and the Th2 cytokines IL-4 and IL-13 are without effect. The ability of TNF-, to up-regulate both TGF-, gene expression and protein release indicates that mast cell,epithelial interactions concerning TGF-, are bi-directional and this may be fundamental to epithelial immunoregulation. The availability of a model system, such as the RPMI 2650 cells, will enable the early evaluation of future novel and targeted interventions directed toward the aberrant responses of upper airway structural cells. [source] REG1A expression is a prognostic marker in colorectal cancer and associated with peritoneal carcinomatosisINTERNATIONAL JOURNAL OF CANCER, Issue 2 2008Christian Astrosini Abstract By expression profiling of early staged colon carcinomas, we found regenerating islet-derived 1 alpha (REG1A) to be upregulated in patients with an unfavorable clinical outcome. For validation, REG1A expression was quantified in another colorectal cancer (CRC) patient cohort by Taqman PCR. Aside from tumor and normal tissue from 63 nonpretreated CRC patients, 31 mucosa biopsies from healthy individuals as well as 22 adenomas were included in the investigation. REG1A was significantly upregulated in tumor specimens (p < 0.001) and adenoma (p < 0.01) as compared to normal colorectal tissue. REG1A expression in normal peritumoral tissue in turn proved to be significantly elevated compared to mucosa from healthy individuals (p < 0.01). Determination of REG1A expression might be useful for early tumor diagnosis with a sensitivity of 90.6%, and a specificity of 77.9%. REG1A expression was significantly increased in tumors with peritoneal carcinomatosis (p < 0.01). Moreover, REG1A turned out to be a significant predictor of disease-free survival (p < 0.05). In conclusion, we present evidence that REG1A is a molecular marker of prognostic value and is associated with peritoneal carcinomatosis in CRC. REG1A turned out to be already significantly raised in peritumoral normal tissue compared to mucosa from healthy individuals, suggesting a molecular field effect of secreted REG1A. © 2008 Wiley-Liss, Inc. [source] | |||||||||||||