Development of a high resolution melting method based on a novel molecular target for discrimination between Bacillus cereus and Bacillus thuringiensis

Abstract

Delimitation within the Bacillus cereus group is confusing due to the highly similar genetic background of its constituent bacteria. This study aimed to develop a rapid and efficient method for the identification of Bacillus cereus and Bacillus thuringiensis, two closely related species within the B. cereus group. Using average nucleotide identity analysis (ANI) and ribosomal multilocus sequence typing (rMLST), the authenticity of the genomes of B. cereus and B. thuringiensis was determined. Emetic B. cereus and Bacillus bombysepticus were also included to provide novel genomic insights into the boundaries within the B. cereus group. Using pan-genome analysis, ispD, a novel core and single-copy molecular target, was identified for the differentiation between B. cereus and B. thuringiensis. Based on the single nucleotide polymorphism within ispD, a high resolution melting (HRM) method for the determination of B. cereus and B. thuringiensis was developed. This method can not only distinguish B. cereus and B. thuringiensis, but can also separate B. cereus from other foodborne pathogenic bacteria. The detection limit of this method could reach 1 pg of pure genomic DNA and 3.7 × 102 cfu/mL of pure culture. Moreover, this new method could effectively differentiate B. cereus and B. thuringiensis in spiked, mixed, and real food samples. Collectively, the established HRM method can provide a new reference paradigm for the sensitive and specific nucleic acid detection of pathogens with identical genomes.