Development and validation of a rapid TaqMan probe-based real-time PCR assay for detecting five subtypes of vancomycin resistance genes.

OBJECTIVE

This study aimed to develop and validate a multiplex TaqMan probe-based real-time PCR assay for the detection of five common vancomycin resistance genes in clinical isolates, including vanA, vanB, vanC, vanD, and vanM, in order to identify resistance mechanisms and support precise clinical antimicrobial therapy.

RESULTS

The established assay showed good overall analytical performance. The limit of detection (LOD) was 10² copies/μL for vanA and vanM, and 10³ copies/μL for vanB, vanC, and vanD. No specific amplification was observed in non-target strains, vancomycin-susceptible Enterococcus faecium (VSEfm), or the no-template control (NTC). The coefficients of variation (CVs) of Ct values for all targets were <1.0% in both intra-assay and inter-assay evaluations. Using NGS as the reference standard, the assay showed an overall agreement of 100.00% (108/108) among the 108 clinical isolates.

CONCLUSION

The multiplex real-time PCR assay established in this study demonstrated good sensitivity, specificity, and repeatability, as well as high concordance with NGS results. Its analytical performance meets the requirements for clinical application. This assay can be used for the rapid detection of five common vancomycin resistance genes and for resistance mechanism typing, thereby providing a useful reference for clinical laboratories in identifying relevant resistance mechanisms and supporting precise antimicrobial therapy.