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Applied and Environmental Microbiology, October 2009, p. 6457-6461, Vol. 75, No. 20
0099-2240/09/$08.00+0     doi:10.1128/AEM.00805-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Multiplex PCR for Detection of Botulinum Neurotoxin-Producing Clostridia in Clinical, Food, and Environmental Samples{triangledown}

Dario De Medici,1* Fabrizio Anniballi,1 Gary M. Wyatt,2 Miia Lindström,3 Ute Messelhäußer,4 Clare F. Aldus,2 Elisabetta Delibato,1 Hannu Korkeala,3 Michael W. Peck,2 and Lucia Fenicia1

Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy,1 Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom,2 Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland,3 Bavarian Health and Food Safety Authority, Oberschleißeim, Germany4

Received 9 April 2009/ Accepted 9 August 2009

Botulinum neurotoxin (BoNT), the most toxic substance known, is produced by the spore-forming bacterium Clostridium botulinum and, in rare cases, also by some strains of Clostridium butyricum and Clostridium baratii. The standard procedure for definitive detection of BoNT-producing clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (SMB). The SMB is highly sensitive and specific, but it is expensive and time-consuming and there are ethical concerns due to use of laboratory animals. PCR provides a rapid alternative for initial screening for BoNT-producing clostridia. In this study, a previously described multiplex PCR assay was modified to detect all type A, B, E, and F neurotoxin genes in isolated strains and in clinical, food, environmental samples. This assay includes an internal amplification control. The effectiveness of the multiplex PCR method for detecting clostridia possessing type A, B, E, and F neurotoxin genes was evaluated by direct comparison with the SMB. This method showed 100% inclusivity and 100% exclusivity when 182 BoNT-producing clostridia and 21 other bacterial strains were used. The relative accuracy of the multiplex PCR and SMB was evaluated using 532 clinical, food, and environmental samples and was estimated to be 99.2%. The multiplex PCR was also used to investigate 110 freshly collected food and environmental samples, and 4 of the 110 samples (3.6%) were positive for BoNT-encoding genes.

* Corresponding author. Mailing address: Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39 06 4990 2477. Fax: 39 06 4990 2045. E-mail: dario.demedici{at}iss.it

{triangledown} Published ahead of print on 14 August 2009.

Applied and Environmental Microbiology, October 2009, p. 6457-6461, Vol. 75, No. 20
0099-2240/09/$08.00+0     doi:10.1128/AEM.00805-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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