Development of a multiplex recombinase polymerase amplification combined with a DNA strip for the rapid detection of...

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).

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.

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.

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.