Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis,Enterococcus faecium and their vanA and vanB genotypes

Background and objectives: we developed and evaluated the utility of a quadruplex taqman real-time pcr assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci.materials and methods: t he s pecificity o f t he a ssay w as t ested u sing r eference s trains o f v ancomycin-resistant a nd susceptible enterococci. in total,193 clinical isolates were identified and subsequently genotyped using a quadruplex taqman real-time pcr assay and melting curve analysis. representative quadruplex taqman real-time pcr amplification curve were obtained for enterococcus faecium,enterococcus faecalis,vana-containing e. faecium,vanb-containing e. faecalis.results: phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates,while in 5 isolates,they were inconsistent. we had three mixed strains,which were detected by the taqman real-time pcr assay and could not be identified correctly using phenotypic methods.conclusion: vancomycin resistant enterococci (vre) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using taqman real-time multiplex real-time pcr assay. © 2014,tehran university of medical science. all rights reserved