Rapid Detection of Bacillus subtilis via RPA Combined with CRISPR/Cas12a

Bacillus and Paenibacillus species are common and widely distributed microorganisms in food systems, often implicated in food spoilage and quality issues. Bacillus subtilis, in particular, has been associated with gas production and package bulging in seasoned foods. In this study, we developed a rapid and visual detection method for Bacillus subtilis by integrating (Recombinase Polymerase Amplification) RPA with (Clustered Regularly Interspaced Short Palindromic Repeats) CRISPR/Cas12a technology (designated as RPA-CRISPR/Cas12a). Specific RPA primers and probes were designed based on the conserved gyrB gene of Bacillus subtilis. Two sets of crRNA were designed according to the number of T-rich PAM sites on the RPA-amplified target sequence, and the reaction conditions were optimized in combination with the CRISPR/Cas12a trans-cleavage detection technology. Under optimized conditions, the crRNA3 guide (with a TT-rich PAM site) demonstrated superior cleavage efficiency compared to crRNA2 (TTT-rich PAM), while crRNA1 (TTTT-rich PAM) showed no activity. The assay achieved a detection limit of 150 pg/μL for genomic DNA and 5.5 CFU/mL for bacterial suspensions within 10 min at 37 °C. The method exhibited high specificity and sensitivity, providing a robust tool for early and on-site detection of Bacillus subtilis in food products.