Cloning and Nucleotide Sequence Analysis of gyrB of Bacillus cereus , B. thuringiensis , B. mycoides , and B. anthracis and Their Application to the Detection of B. cereus in Rice

Abstract

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 ofB. cereus JCM 2152T,Bacillus thuringiensis IAM 12077T,Bacillus mycoides ATCC 6462T, andBacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from withingyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, andB. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, 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 amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.

Phenotypic and genotypic heterogeneity among closely related soil-borne N2- and N2O-producing Bacillus isolates harboring the nosZ gene

“…This differs from the clustering observed in the 16S rRNA gene phylogeny, where ULT-42 showed little differentiation from other isolates. This discrepancy is not surprising, considering that the 16S rRNA gene is not optimal for discriminating among Bacillus strains (Yamada et al, 1999). Previous studies have also shown that Bacillus species may harbor multiple copies of ribosomal genes, which may vary up to 2% (Acinas et al, 2004).…”

Rapid genotypic detection ofBacillus anthracisand theBacillus cereusgroup by multiplex real-time PCR melting curve analysis

“…anthracis is a highly fatal infectious agent in animals and humans and therefore its early and unambiguous diagnostic detection is essential for successful treatment and disease prevention. There have been many efforts to utilize rapid DNA-based detection methods, such as PCR [22][23][24][25][26][27][28][29][30][31][32][33], to replace time-consuming biochemical or culture-based diagnostic tests [34]. PCR-based methods can readily differentiate vaccine or fully virulent B. anthracis plasmid genotypes [22][23][24]26,35].…”

“…PCR methods developed for detection of the B. anthracis chromosome have suffered from lack of assay specificity; Ba813 [25,26,51], vrrA gene [27][28][29], gyrase B gene (gyrB) [30], SG-850 [31], the b subunit of RNA Table 1 Bacterial strains used in this study and their DNA-based assay response (manuscript in preparation)…”

Polymerase chain reaction assay for the detection of Bacillus cereus group cells

“…Yamada et al [16] and Hsieh et al [7] also developed primer sets which can be used for the speci¢c detection of B. cereus group cells. These primers were directed against genes for a cold-shock protein and a sphingomyelinase, respectively.…”