Development of a multiplex real-time PCR assay for the identification and quantification of group-specific Bacillus...

Spoilage microorganisms can occur at many points throughout food production systems. Bacillus spp. and Paenibacillus spp. are important aerobic spoilage bacteria in various sectors of the food industry. In this study, we developed a rapid detection and quantification technique for Bacillus group-specific and the genus Paenibacillus by using multiplex quantitative PCR (mqPCR). The 1st was the Bacillus cereus group containing B. cereus and B. weihenstephanensis; the 2nd was the B. subtilis group containing B. subtilis, B. licheniformis, B. safensis, and B. pumilus; the 3rd was the B. simplex group containing B. megaterium and B. simplex; and the 4th was the genus Paenibacillus. Depending on the assays, the detection limit was 10 copy numbers. In addition, mqPCR assays were validated by spiking potato salad and milk samples with four strains; B. weihenstephanensis, B. licheniformis, B. megaterium, and P. lautus. The detection dynamic range for potato salad was 10⁵ CFU/mL-10¹ CFU/mL with B. weihenstephanensis and B. licheniformis, and 10⁵ CFU/mL-10² CFU/mL with B. megaterium and P. lautus, while, for milk, all strains were 10⁵ CFU/mL-10² CFU/mL. We also stored these food matrices spiked with four bacterial suspensions (approximately 10³ CFU/mL) at various temperatures. Results showed that B. weihenstephanensis and B. licheniformis were able to grow in potato salad, whereas, the populations of B. weihenstephanensis, B. licheniformis, and P. lautus increased in milk. Consequently, the mqPCR assays developed here in facilitated the differentiation, quantification, and confirmation of the presence of the psychrophilic and psychrotolerant Bacillus group and Paenibacillus spp.