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

Area of Science:

• Molecular Biology
• Diagnostic Assays
• Microbiology

Background:

• Enterococcus faecalis is a significant opportunistic pathogen causing severe healthcare-associated infections (HAIs).
• Rising multidrug resistance in E. faecalis necessitates rapid and accurate diagnostic methods.
• Current detection methods can be time-consuming and require specialized laboratory equipment.

Purpose of the Study:

• To develop a rapid, sensitive, and specific detection assay for Enterococcus faecalis.
• To utilize the CRISPR/Cas12a system combined with recombinase polymerase amplification (RPA) for pathogen identification.
• To establish a cost-effective and user-friendly diagnostic tool suitable for clinical settings.

Main Methods:

• Targeted amplification of the E. faecalis pheS gene using recombinase polymerase amplification (RPA).
• CRISPR/Cas12a system employed for specific recognition of the amplified DNA.
• Detection of CRISPR/Cas12a activity via collateral ssDNA cleavage, generating a quantifiable fluorescent signal.

Main Results:

• The assay achieved a limit of detection (LOD) of 10^-2 ng/μL with high specificity.
• No cross-reactivity was observed with other common pathogens.
• Results from clinical isolates and spiked samples demonstrated high reliability, consistent with PCR/qPCR methods.

Conclusions:

• The developed RPA-CRISPR/Cas12a assay provides a sensitive, specific, and reliable method for E. faecalis detection.
• The assay's simplicity, minimal equipment needs, and cost-effectiveness make it suitable for rapid clinical diagnostics.
• This method holds promise for improving diagnostic efficiency and supporting antimicrobial stewardship, particularly in resource-limited environments.