Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB.

The aim of the study was to identify and evaluate specific biomarkers to differentiate within Bacillus cereus group species from contaminated food samples with the use of real-time PCR. A total of 120 strains, comprising of 28 reference, 2 type, 78 wild strains of B. cereus and B. thuringiensis along with 12 strains representing 2 bacterial groups - B. mycoides, B. pseudomycoides, B. weihenstephanensis (B. cereus group); B. amyloliquefaciens, B. subtilis, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Micrococcus luteus, Salmonella enterica, Staphylococcus aureus, Streptococcus pyogenes (non-Bacillus sp.) were identified by applying valid biomarkers (groEL and gyrB). In addition, the presence of B. cereus group was determined in three different artificially contaminated vegetable samples (lettuce, spinach, and kimbap), using prominent biomarkers targeting on chaperonin protein (GroEL) and topoisomerase enzyme protein (gyrB). Direct analysis of samples revealed the specificity towards identification and characterization of the B. cereus group among wild, reference and type strains and the type strain inoculated in vegetables. Our results demonstrated two existing biomarkers groEL and gyrB with a high specificity of 98% and 96% respectively to analyze the total B. cereus group. Further, we also reported the detection limit of groEL and gyrB in food samples was 3.5 and 3.7 log CFU/g respectively. Thus, the developed real-time PCR approach can be a reliable and effective tool for the identification of B. cereus group strains present in environment and food samples. This does not require band isolation, re-amplification, sequencing or sequence identification, thus reducing the time and cost of analysis.