Development of a multiplex recombinase polymerase amplification combined with a DNA strip for the rapid detection of <i>Enterococcus faecium</i> , <i>Enterococcus faecalis</i> , and vancomycin resistance genes

Background Vancomycin-resistant enterococci (VRE), particularly Enterococcus faecium and E. faecalis , are responsible for various human infections and represent a significant global public health concern in both nosocomial and community settings. To facilitate early detection of these pathogens, we developed a rapid method for detecting E. faecium , E. faecalis , and vancomycin resistance genes ( vanA and vanB ) in a single reaction using multiplex recombinase polymerase amplification (M-RPA) combined with a chromatographic printed-array strip (C-PAS). Methods A total of 106 samples, including 46 E. faecium (35 vanA -type and one vanB -type strains), 39 E. faecalis (one vanA -type and two vanB -type strains), four Enterococcus spp., and 17 non- Enterococcus spp. isolates were tested using the M-RPA-C-PAS method and compared with the conventional polymerase chain reaction (PCR) method. Specific tagged and biotinylated M-RPA primers were designed for detecting ddl ( E. faecium ), sodA ( E. faecalis ), vanA , and vanB . The M-RPA reaction was performed under isothermal conditions at 37 °C for 20 min, and visual detection using the C-PAS for DNA signal detection within another 20 min. The performance of the M-RPA-C-PAS was initially evaluated in 12 spiked rectal swabs and 12 positive blood culture samples from patients compared with the PCR method. Results Compared with the PCR-based method, the M-RPA-C-PAS method demonstrated sensitivities of 95.7%, 94.9%, and 100.0% and specificities of 100.0%, 92.5%, and 100.0% for ddl , sodA , and vanA , respectively, and 93.2% specificity for vanB . The lower detection limits of the M-RPA-C-PAS method for detecting the ddl and sodA genes were 10⁴ and 10³ CFU/mL, respectively, whereas those of the PCR method were 10⁵ and 10⁴ CFU/mL, respectively. The lower limit of detection of both methods for vanA and vanB was 10⁴ CFU/mL. The M-RPA-C-PAS assay showed 100% agreement with PCR for identifying the four genes in the spiked and clinical samples. Conclusion The M-RPA-C-PAS method is an alternative rapid approach with high sensitivity and specificity, demonstrating comparable performance to conventional PCR. Therefore, the M-RPA-C-PAS method represents a promising point-of-care diagnostic tool for the rapid detection of VRE, supporting timely clinical management and infection control strategies.