Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B-thuringinesis, B-mycoides, and B-anthracis and their application to the detection of B-cereus in rice

As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B, cereus J CM 2152(T), Bacillus thuringiensis IAM 12077(T), Bacillus mycoides ATCC 646 2(T), and Bacillus anthracis Pasteur #2H were cloned and sequenced, Oligonu cleotide PCR primer sets were designed from within gyrB sequences of the re spective bacteria for the specific amplification and differentiation of B. cereus, B, thuringiensis, and B, anthracis. The results from the amplificat ion of gyrB sequences correlated well,vith results obtained with the 16S rD NA-based hybridization study but not with the results of their phenotypic c haracterization. Some of the reference strains of both B. cereus (three ser ovars) and B. thuringiensis (two serovars) were not positive in PCR amplifi cation assays with gyrB primers. However, complete sequencing of 1.2-kb gyr B fragments of these reference strains showed that these serovars had, in f act, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplificat ion of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B, cercus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration st ep is required to remove PCR inhibitory substances.