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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

Yamada, Shoichi and Ohashi, Eiji and Agata, Norio and Venkateswaran, Kasthuri (1999) 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. Applied and Environmental Microbiology, 65 (4). pp. 1483-1490. ISSN 0099-2240. http://resolver.caltech.edu/CaltechAUTHORS:YAMaem99

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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 of B. cereus JCM 2152T, Bacillus thuringiensis IAM 12077T, Bacillus mycoides ATCC 6462T, and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. 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.


Item Type:Article
Additional Information:Copyright © 1999, American Society for Microbiology. Received 25 September 1998/Accepted 5 January 1999 We are grateful to M. Satake for encouragement, I. Uchida for providing DNA of B. anthracis, and I. Sugahara for various Bacillus strains. We also thank Y. Kamijoh, T. Kurusu, K. Hanai, and Y. Hara for their technical assistance.
Record Number:CaltechAUTHORS:YAMaem99
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:YAMaem99
Alternative URL:http://aem.asm.org/cgi/content/abstract/65/4/1483
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ID Code:2814
Collection:CaltechAUTHORS
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Deposited On:28 Apr 2006
Last Modified:26 Dec 2012 08:51

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