Caracterização de fatores de virulência presentes em isolados de Escherichia coli provenientes da região Sul do Brasil

Foram analisados neste trabalho 42 isolados de Escherichia coli originários de 15 granjas de suínos da regiãoSul do Brasil. Os isolados foram obtidos a partir de fezes de suínos com suspeita de colibacilose. As amostras foramcaracterizadas quanto a presença de fatores de virulência (FVs) pela reação em cadeia da polimerase (PCR), queconsistiu na detecção das fímbrias F4, F5, F6, F18 e F41 e das toxinas Stb, StaP, Stx2e e LT. Dos 42 isolados,somente 15 foram confirmados como portadores dos FVs estudados, sendo que as toxinas mais freqüentementeencontradas foram Stb seguida da StaP. Nos isolados portadores de toxinas foram encontradas as fímbrias F18, F4 e F5, e 4 isolados toxigênicos não apresentaram as fímbrias analisadas. A utilização da PCR para detecção detoxinas e fímbrias demonstrou ser um teste prático no diagnóstico da colibacilose.Palavras-chave: Escherichia coli, ETEC, PCR, colibacilose, fatores de virulência

A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping