Development of an ultrafast PCR to detect clinically relevant acquired vancomycin-resistance genes from cultured enterococci.

ABSTRACT

BACKGROUND: VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA, vanB, vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. METHODS: Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. RESULTS: All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). CONCLUSIONS: The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE.