Bacillus cereus group strains, their hemolysin BL activity, and their detection in foods using a 16S RNA and hemolysin BL gene-targeted multiplex polymerase chain reaction system

Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group st rains. It is also a target enterotoxin for the most commonly used B. cereus detection kit, i.e., the B. cereus enterotoxin (diarrheal type) reversed p assive latex agglutination (BCET-RPLA) test kit. A survey of the HBL activi ties and the cytotoxicities to the Chinese hamster ovary (CHO) cells for th e B. cereus group strains, however, showed that although only part of the B . cereus group strains are HBL active, all strains show cytotoxicity to the CHO cells. Thus, methods that allow the detection of not only the HBL but also of the B, cereus group strains are important. In this study, by compar ison of the gene sequences of the 16S rRNA for B. cereus group and other ba cteria strains, we designed primers B16S1 and B16S2 specific to all the B. cereus group strains. In addition, because HBL is a major enterotoxin, we a lso designed HBL gene-specific polymerase chain reaction (PCR) primers, i.e ., Hm1 and Hm2, that generated the same results as those of the hemolysis a nd BCET-RPLA assays. Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B. cereus group c ells and the possible presence of their HBL enterotoxins. Also, all these P CR systems allowed the detection of n x 10(0) CFU B. cereus cells per g of food sample if an 8-h enrichment step was performed prior to the PCR.