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Applied and Environmental Microbiology, March 2005, p. 1148-1154, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1148-1154.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Efficient DNA Fingerprinting of Clostridium botulinum Types A, B, E, and F by Amplified Fragment Length Polymorphism Analysis

Riikka Keto-Timonen,* Mari Nevas, and Hannu Korkeala

Department of Food and Environmental Hygiene, University of Helsinki, Helsinki, Finland

Received 3 June 2004/ Accepted 11 October 2004

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.

* Corresponding author. Mailing address: Department of Food and Environmental Hygiene, P.O. Box 66, FI-00014 University of Helsinki, Helsinki, Finland. Phone: 358-9-19157145. Fax: 358-9-19157101. E-mail: riikka.keto-timonen{at}helsinki.fi.

Applied and Environmental Microbiology, March 2005, p. 1148-1154, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1148-1154.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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