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Applied and Environmental Microbiology, December 2001, p. 5694-5699, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5694-5699.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

Miia Lindström,* Riikka Keto, Annukka Markkula, Mari Nevas, Sebastian Hielm, and Hannu Korkeala

Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, FIN-00014 Helsinki University, Finland

Received 21 May 2001/Accepted 22 September 2001

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102 cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10-2 spore/g for types A, B, and F to 10-1 spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.

* Corresponding author. Mailing address: Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, P.O. Box 57, FIN-00014 Helsinki University, Finland. Phone: 358-9-191 49702. Fax: 358-9-191 49718. E-mail: mklindst{at}mappi.helsinki.fi.

Applied and Environmental Microbiology, December 2001, p. 5694-5699, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5694-5699.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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