Simultaneous and Accurate Visual Detection of Vancomycin-Resistant Enterococci vanA, vanB and vanM by Multiplex Recombinase Polymerase Amplification Combined with Lateral Flow Strip.

Vancomycin-resistant Enterococcus (VRE) has demonstrated increasing global prevalence in recent years. Clinical detection currently relies on phenotypic methods including agar screening, minimum inhibitory concentration (MIC) testing, Kirby-Bauer disk diffusion, and Etest. In addition, molecular approaches such as polymerase chain reaction (PCR) and quantitative PCR (qPCR) can be applied for VRE identification. Nevertheless, these methods cannot achieve point-of-care detection (POCT). Thus, novel rapid diagnostic platforms have become urgently needed for curbing VRE transmission and containing nosocomial outbreaks. Recombinase polymerase amplification (RPA) and lateral flow strips (LFS) are effective tools for achieving rapid POCT. In this study, RPA was combined with LFS to establish a fast, sensitive, and specific detection method. This study established a multiplex RPA-LFS (mRPA-LFS) that delivers results within 30-40 min, with detection limits of 102 copies/μl for vanA, vanB, and vanM. Notably, the assay demonstrated high specificity without cross-reactivity to common bacterial/fungal pathogens, and showed 100% concordance with conventional PCR in 30 clinical samples. In this study, a rapid detection assay for vanA, vanB, and vanM genes in VRE was developed using mRPA-LFS technology. Characterized by high sensitivity, specificity, operational simplicity, and cost-effectiveness, this method is suitable for on-site detection.