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Real-time PCR Amplification (real-time + pcr_amplification)
Selected AbstractsRapid diversification of measles virus genotypes circulating in Morocco during 2004,2005 epidemics,JOURNAL OF MEDICAL VIROLOGY, Issue 11 2006Amal Alla Abstract Measles virus strains circulating in six different regions in Morocco during 2004,2005 were analysed. They were genotyped using two different methods: the recently developed method based on real-time PCR amplification and melting curve analyses, and the conventional method based on nucleic acid sequencing and phylogenetic analysis of 456 nucleotides of the 3,-region of the nucleoprotein (N) gene sequence. Five genotypes (A, B3.2, C2, D7 and D8) were shown to be circulating during this period. Previous studies on measles virus genotypes in Morocco (1998,2003) showed that only the genotype C2 was present and was considered to be endemic. Sequence comparison of the 2004,2005 viruses with other measles strains suggests that measles strains belonging to genotype B3.2 were probably imported from West Africa, whereas those belonging to genotypes D7 and D8 were imported from Europe. These studies which identify the route of importation of measles are important for developing strategies for measles elimination in Morocco. J. Med. Virol. 78:1465,1472, 2006. © 2006 Wiley-Liss, Inc. [source] Detection of Leishmania infantum by real-time PCR in a canine blood bankJOURNAL OF SMALL ANIMAL PRACTICE, Issue 7 2008M. D. Tabar Objectives: Risk for transmission of Leishmania infantum from blood products has been largely demonstrated in human and veterinary literature. Appropriate screening of canine blood donors is important especially in an endemic area such as Barcelona (Spain). The purpose of this study was to evaluate the presence of L infantum DNA parasites by real-time quantitative PCR in our canine blood bank. Methods: Samples from blood products obtained from 92 canine blood donors were assayed for L infantum by means of real-time PCR amplification and quantification. Results: The prevalence of quantitative PCR-positive blood samples among healthy seronegative blood donors was 19·6 per cent. Clinical Significance: The results of this study show that L infantum infection is common in canine blood donors and their blood products in an endemic area, despite a negative commercial serological screening for infectious diseases. Therefore, screening by PCR should be included in an integrated approach to evaluate L infantum infection among potential blood donors. [source] Gene expression profiles of TNF-,, TACE, furin, IL-1, and matrilysin in UVA- and UVB-irradiated HaCat cellsPHOTODERMATOLOGY, PHOTOIMMUNOLOGY & PHOTOMEDICINE, Issue 4 2005Beata Skiba Background/Purpose: It is known that solar ultraviolet (UV) irradiation exerts multiple effects on mammalian skin tissues, one of which is the induction of local and systemic immunosuppression as well as inflammation. Tumor necrosis factor-, (TNF-,) and other cytokines are suggested to play a role in these responses. Quantitative real-time polymerase chain reaction (TaqMan RTPCR) was used to elucidate the effect of UVA and UVB irradiation on the expression of genes coding for TNF-,, IL-1,, IL-10, FasL, matrilysin, TACE and furin in HaCaT cells over a 48 h period (IL-1,, interleukin-1,; FasL, Fas ligand). Methods: Cultured HaCaT cells were either sham irradiated (control) or exposed to UVA (2000 and 8000 J/m2) or UVB (200 and 2000 J/m2) radiation. RNA was extracted from cells at 0, 4, 8, 12, 16, 24, 48 h post-irradiation and reverse transcribed to generate cDNA for subsequent real-time PCR amplification. Results: Significant increases in the mRNA levels for all genes tested were detected in both UVA- and UVB-irradiated HaCaT cells compared with control (sham-irradiated) cells. TNF-, mRNA levels were immediately up-regulated (0 h) after irradiation, with maximal induction at 8 h post 2000 J/m2 UVA and 200 J/m2 UVB irradiation, at 4 h post 8000 J UVA irradiation and at 48 h post 2000 J/m2 UVB irradiation. No correlation was observed between TNF-,, TACE and furin mRNA induction in the different irradiated cohorts. Conclusion: Results suggest that time-distinct gene induction of TNF-,, furin, IL-1, and matrilysin may be involved in UV-induced cellular responses, but not for TACE. In general, mRNA induction was dose dependent at some time points post-irradiation, but not throughout the whole time course tested. Our results show that quantitative real-time PCR is a useful tool in the analysis of quantitative changes of mRNA levels in cultured HaCaT cells after UV exposure. [source] Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b genePLANT PATHOLOGY, Issue 1 2009S. Banno Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene (cytb) was characterized. All QoI-resistant isolates had the same mutation (GGT to GCT) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b, which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real-time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI-resistant and QoI-sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI-resistant and QoI-sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild-type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI-sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi-143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi-143/144 intron were azoxystrobin-sensitive, suggesting that the QoI-resistant mutation at codon 143 in cytb prevents self-splicing of the Bcbi-143/144 intron, as proposed in some other plant pathogens. [source] | |||||||||||||