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Development of multiplex recombinase polymerase amplification combined with DNA strip for rapid detection of Enterococcus faecium, Enterococcus faecalis and vancomycin resistance genes

microbiology
molecular-biology
infectious-diseases

Abstract

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, 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 M - RPA tagged and biotinylated primers were designed for the ddl ( E . faecium ) , sodA ( E . faecalis ) , vanA, and vanB genes . The M - RPA reaction was performed under isothermal conditions at 37°C for 20 minutes and visual detection using the C - PAS for DNA signal detection within an additional 20 minutes .
Results: The M - RPA - C - PAS method demonstrated sensitivities of 95 . 7 % , 94 . 9 % , 100.0 % and 100.0 % ; and specificities of 100.0 % , 92 . 5 % , 100.0 % , and 93 . 2 % for ddl, sodA, vanA, and vanB genes, respectively, compared with the PCR methods . The lower detection limits of the M - RPA - C - PAS method for detecting ddl, sodA, vanA, and vanB genes were 10⁴, 10³, 10⁴, and 10⁴ CFU / mL, respectively, whereas those of the PCR method were 10⁵, 10⁴, 10⁴, and 10⁴ CFU / mL, respectively .
Conclusion: The M - RPA - C - PAS method is an alternative rapid approach with high sensitivity and specificity, demonstrating performance comparable to conventional PCR . This method represents a promising point - of - care diagnostic tool for the rapid detection of VRE, supporting timely clinical management and infection control strategies .
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