Development and application of a rapid dual RPA-LFS assay for simultaneous detection of Enterococcus faecalis and Enterococcus faecium.

Enterococcus faecium and Enterococcus faecalis, ranked among the top three global nosocomial pathogens (WHO data), pose escalating threats due to multidrug resistance (particularly vancomycin-resistant strains, VRE) and foodborne transmission. Conventional detection relies on time-consuming culture-based typing (ISO 7899-2; 48-72 h) with > 12% false-negative rates, while molecular methods like qPCR require sophisticated instrumentation, limiting field deployment. This study developed a rapid dual recombinase polymerase amplification-lateral flow strip (RPA-LFS) assay for simultaneous detection of E. faecium and E. faecalis, addressing critical bottlenecks of prolonged turnaround time, species differentiation challenges, and equipment dependency. Species-specific primers and probes were designed targeting conserved genomic regions. Probes featured 5'-FITC/DIG reporter groups and amplification products incorporated 3'-biotin modifications, enabling THF cleavage-mediated signal release. Following optimization (37 °C, 20 min), results were visually interpreted via LFS within 10 min. Key findings include: Sensitivity: 10 CFU/mL (E. faecalis) and 102 CFU/mL (E. faecium), maintained in complex matrices; Specificity: 100% (no cross-reactivity with 30 non-target strains); Clinical validation: 100% sensitivity and specificity (n = 435 sputum isolates), significantly outperforming culture methods. This assay achieves equipment-free species differentiation within 40 min, providing a transformative tool for precision antimicrobial therapy, food safety monitoring, and frontline antimicrobial resistance (AMR) containment.