Application of comparative genomics in the development of DNA probes to detect Bacillus cereus and Bacillus subtilis

Abstract Primer-probe sets to detect Bacillus cereus and/or B. subtilis in food products were developed based on panX (a pan-genome analysis tool). Data for 60 B. cereus strains and 131 B. subtilis strains retrieved from the NCBI RefSeq database were analyzed by panX. Primer-probe sets for B. cereus (ccpA) and B. subtilis (cotQ) were developed by comparing the core genomes of the two bacteria. The developed primer-probe sets’ sensitivity and selectivity were identified using 45 strains, including ten B. cereus, five B. subtilis, 13 non-B. cereus and non-B. subtilis strains, and 17 non-Bacillus strains using real-time polymerase chain reaction (PCR). The developed primer-probe sets displayed high sensitivity and selectivity for B. cereus and B. subtilis, validated with two different real-time PCR instruments. PCR analysis using the developed primer-probe showed high efficiency in buffer and milk samples. These results indicate that the primer-probe sets developed based on the panX analysis can be used effectively for detecting B. cereus and/or B. subtilis in food products.