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A Unique Restriction Site in the flaA Gene Allows Rapid Differentiation of Group I and Group II Clostridium botulinum Strains by PCR–Restriction Fragment Length Polymorphism Analysis

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Abstract:

Clostridium botulinum produces the potent botulinum neurotoxin, the causative agent of botulism. Based on distinctive physiological traits, strains of C. botulinum can be divided into four groups: however, only groups I and II are associated with human illness. Alignment of the flaA gene sequences from 40 group I and 40 group II strains identified a single Bsr G1 restriction cut site that was present at base pair 283 in all group II flaA sequences and was not found in any group I sequence. The flaA gene was amplified by rapid colony PCR from 22 group I strains and 18 group II strains and digested with Bsr GI restriction enzyme. Standard agarose gel electrophoresis with ethidium bromide staining showed two fragments, following restriction digestion of group II flaA gene amplicons with Bsr GI, but only a single band of uncut flaA from group I strains. Combining rapid colony PCR with Bsr GI restriction digest of the flaA gene at 60°C is a significant improvement over current methods, such as meat digestion or amplified fragment length polymorphism, as a strain can be identified as either group I or group II in under 5 h when starting with a visible plated C. botulinum colony.

Document Type: Research Article

Affiliations: Bureau of Microbial Hazards, Health Products and Food Branch, Health Canada, Sir Frederick G. Banting Research Centre, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, Canada K0A 0K9

Publication date: September 1, 2007

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