A novel method of species-specific molecular target mining and accurate discrimination of Bacillus cereus sensu lato

Bacillus cereus, a member of the Bacillus cereus sensu lato (B. cereus s.l.), is widely distributed in nature and can contaminate a variety of foods, leading to foodborne illnesses and substantial losses in the food industry. Although culture-based methods remain the gold standard for identifying B. cereus due to their high sensitivity under specific conditions, they are often complex and labor-intensive to implement. Furthermore, the high genetic similarity among certain members of the B. cereus s.l. makes it challenging to identify species-specific molecular targets, hindering the rapid and accurate differentiation of these bacteria. In this study, we introduce a novel method, comparative analysis based on whole genome slices (CAWGS), combined with the Basic Local Alignment Search Tool (BLAST) for efficient molecular target mining. Using CAWGS-BLAST and pan-genome analysis, we successfully identified new molecular targets for B. cereus, Bacillus thuringiensis, emetic B. cereus, Bacillus anthracis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus megaterium. Based on these newly discovered targets, we developed a PCR-CRISPR/Cas12a method for detecting B. cereus s.l. and related species. Our research not only provides a rapid and accurate approach for discriminating B. cereus s.l. and related species, but also offers a universal and valuable reference for detecting foodborne pathogens, especially those with highly similar phenotypic and genetic characteristics.