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
S. Yamada et al., 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, APPL ENVIR, 65(4), 1999, pp. 1483-1490
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.