Identification of a Bacillus anthracis specific indel in the yeaC gene and development of a rapid pyrosequencing assay for distinguishing B. anthracis from the B. cereus group |
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| Institution: | 1. Department for Bioanalysis and Horizon Technologies, Centre for Infections, Health Protection Agency, 61 Colindale Avenue, Colindale, London, NW9 5EQ United Kingdom;2. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Canada L8N 3Z5;1. Karadeniz Technical University, Faculty of Sciences, Department of Molecular Biology and Genetic, 61080 Trabzon, Turkey;2. Karadeniz Technical University, Faculty of Sciences, Department of Biology, 61080 Trabzon, Turkey;3. Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste. Marie, Ontario, Canada;1. Department of Food Engineering, Faculty of Engineering, Bayburt University, Bayburt, Turkey;2. Analytical Sciences Unit, Quadram Institute Bioscience, Norwich Research Park, Norwich NR4 7UA, UK;3. Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, IL 61604, USA;4. Gut Health and Food Safety Programme, Quadram Institute Bioscience, Norwich NR4 7UA, UK;1. Department of Wine, Food and Molecular Biosciences, Lincoln University, Springs Road, Lincoln 7467, New Zealand;2. US Department of Agriculture, Produce Safety and Microbiology Research Unit, Albany, CA, USA;3. Bioinformatics and Statistics Group, School of Fundamental Sciences, Massey University, Palmerston North, New Zealand;4. mEpiLab, School of Veterinary Science, Massey University, Palmerston North, New Zealand;5. Institute of Environmental Science and Research, Christchurch, New Zealand;6. Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium |
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| Abstract: | Bacillus anthracis, the causative agent of anthrax, is a potential source of bioterrorism. The existing assays for its identification lack specificity due to the close genetic relationship it exhibits to other members of the B. cereus group. Our comparative analyses of protein sequences from Bacillus species have identified a 24 amino acid deletion in a conserved region of the YeaC protein that is uniquely present in B. anthracis. PCR primers based on conserved regions flanking this indel in the Bacillus cereus group of species (viz. Bacillus cereus, B. anthracis, B. thuringiensis, B. mycoides, B. weihenstephnensis and B. pseudomycoides) specifically amplified a 282 bp fragment from all six reference B. anthracis strains, whereas a 354 bp fragment was amplified from 15 other B. cereus group of species/strains. These fragments, due to large size difference, are readily distinguished by means of agarose gel electrophoresis. In contrast to the B. cereus group, no PCR amplification was observed with any of the non-B. cereus group of species/strains. This indel was also used for developing a rapid pyrosequencing assay for the identification of B. anthracis. Its performance was evaluated by examining the presence or absence of this indel in a panel of 81 B. cereus-like isolates from various sources that included 39 B. anthracis strains. Based upon the sequence data from the pyrograms, the yeaC indel was found to be a distinctive characteristic of various B. anthracis strains tested and not found in any other species/strains from these samples. Therefore, this B. anthracis specific indel provides a robust and highly-specific chromosomal marker for the identification of this high-risk pathogen from other members of the B. cereus group independent of a strain's virulence. The pyrosequencing platform also allows for the rapid and simultaneous screening of multiple samples for the presence of this B. anthracis-specific marker. |
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