Rapid detection of Enterococcus and vancomycin resistance using recombinase polymerase amplification

Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium , have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes ( vanA and vanB ) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium ) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium ; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium , vanA , and vanB , and it has the potential to be used as a point-of-care device for VRE therapy and prevention.