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.
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 BsrG1 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 BsrGI restriction enzyme. Standard agarose gel electrophoresis with ethidium bromide staining showed two fragments, following restriction digestion of group II flaA gene amplicons with BsrGI, but only a single band of uncut flaA from group I strains. Combining rapid colony PCR with BsrGI restriction digest of the flaA gene at 60 degrees 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.
Paul
Catherine J
CJ
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.
Tran
Shulin
S
Tam
Kevin J
KJ
Austin
John W
JW
D003014:Clostridium botulinum / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis; D005408:Flagellin / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D005408:Flagellin; C067138:flaA protein, bacteria