To establish a rapid detection method for Clostridium botulinum in food.

A rapid detection method based on RPA-CRISPR/Cas12a was developed by integrating recombinase polymerase amplification (RPA) with the CRISPR-Cas12a system. After the reaction conditions were optimized, the method’s sensitivity, specificity, and usefulness were methodically confirmed.

The optimized method achieved detection within 1 hour, with a limit of detection (LOD) of 1.91 copies/μL. No cross-reactivity was observed with non-target pathogens. The RPA-CRISPR/Cas12a-based detection method developed in this study exhibits high specificity, sensitivity, and operational simplicity and may provide a feasible solution for the rapid detection of foodborne pathogens.