AEM Accepts, published online ahead of print on 14 August 2009

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Appl. Environ. Microbiol. doi:10.1128/AEM.00805-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Multiplex PCR to detect botulinum neurotoxin-producing clostridia in clinical, food and environmental samples.

Dario De Medici^*, Fabrizio Anniballi, Gary M. Wyatt, Miia Lindström, Ute Messelhäußer, Clare F. Aldus, Elisabetta Delibato, Hannu Korkeala, Michael W. Peck, and Lucia Fenicia

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

^* To whom correspondence should be addressed. Email: dario.demedici{at}iss.it.

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 with 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 published multiplex PCR assay has been modified to detect all type A, B, E and F neurotoxin genes on isolated strains and in clinical, food, environmental samples. The assay includes an internal amplification control. The effectiveness of the multiplex PCR method to detect clostridia possessing types A, B, E and F neurotoxin genes was evaluated by direct comparison with the SMB. The method showed 100% inclusivity and 100% exclusivity using 182 BoNT-producing clostridia and 21 other bacterial strains. The relative accuracy between 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. Four out of 110 (3.6%) were positive for BoNT-encoding genes.