ارزیابی فیلوژنی سویه های اشریشیا کلی جدا شده از بیماران مبتلا به عفونت ادراری در استان بوشهر به روش Real-time PCR

Background and Objectives: Uropathogenic E. coli (UPEC) are among major pathogens causing of 80-90% of all episodes of Urinary tract infections (UTIs). Recently, Escherichia coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F and clade I. This study was aimed to develop a rapid, sensitive and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. Material and Method: This study was carried out on the E. coli strains (isolated from patient with UTI) in which the presence of all seven target genes had confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons having melting temperatures (Tm) ranging from 82°C to 93°C, with more than 1.5°C between each peak. Results: The multiplex real-time PCR assays developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for ArpAgpE and 91±0.5 for internal control. We also confirmed the specific amplifications by detection of the specific bands in electrophoresis. Conclusion: To our knowledge, this study is the first melting curve-based real-time PCR assay developed for the simultaneous and separated detection of these seven target genes. Our findings showed, this assay has the potential to be a rapid, reliable and cost–effective alternative for routine phylo-typing of E. coli strains. Keywords: Escherichia coli, Phylogenetic groups, Eva-Green real-time PCR, Melting curve.