Rapid detection of <i>Enterococcus faecalis</i> using RPA-CRISPR/Cas12a-assisted technology

ABSTRACT Enterococcus faecalis ( E. faecalis ) is an opportunistic pathogen capable of causing various life-threatening infections, including urinary tract infections, bloodstream infections, infective endocarditis, and meningitis. As a major etiological agent of healthcare-associated infections (HAIs), its global prevalence continues to rise, a trend closely linked to the increasing problem of multidrug resistance driven by overuse of antibiotics. Therefore, rapid and accurate detection is essential for timely treatment and improved prognosis. In this study, the pheS gene of E. faecalis was rapidly amplified using recombinase polymerase amplification (RPA), and detection was achieved via a CRISPR/Cas12a system. The Cas12a-crRNA complex specifically recognized the amplification product and triggered nonspecific cleavage of a single-stranded DNA (ssDNA) reporter, generating a fluorescent signal that could be quantified in a real-time PCR system or visualized directly under ultraviolet (UV) light. After optimization of key parameters—including RPA primers, reaction conditions, crRNA sequence, and the crRNA/Cas12a combination—the assay achieved a limit of detection (LOD) of 10 −2 ng/μL within a short turnaround time, and showed no cross-reactivity with other common pathogen detection results from clinical isolates and spiked samples were fully consistent with those obtained through PCR/qPCR, confirming high reliability. In summary, the RPA-CRISPR/Cas12a detection method established in this study is sensitive, specific, and reliable. Its simplicity, minimal equipment requirements, and cost-effectiveness make it a promising tool for rapid clinical detection of E. faecalis . IMPORTANCE Enterococcus faecalis is a major opportunistic pathogen responsible for severe healthcare-associated infections, with rising prevalence linked to antibiotic resistance. Rapid and accurate detection is critical for timely treatment and infection control. Conventional methods are often time-consuming or require complex laboratory infrastructure, limiting their use at the point of care. This study developed a rapid detection assay by integrating recombinase polymerase amplification with the CRISPR/Cas12a system, targeting the pheS gene of E. faecalis . The method is sensitive and specific, providing visual results under UV light within a short turnaround time. It offers a simple, cost-effective, and requires minimal equipment, suitable for clinical and resource-limited settings, potentially improving diagnostic efficiency and supporting antimicrobial stewardship.