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Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum
Article first published online: 9 MAR 2009
DOI: 10.1111/j.1365-2672.2009.04215.x
© 2009 The Authors. Journal compilation © 2009 The Society for Applied Microbiology
Additional Information
How to Cite
Fach, P., Micheau, P., Mazuet, C., Perelle, S. and Popoff, M. (2009), Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum. Journal of Applied Microbiology, 107: 465–473. doi: 10.1111/j.1365-2672.2009.04215.x
Publication History
- Issue published online: 9 JUL 2009
- Article first published online: 9 MAR 2009
- 2008/1051: received 23 June 2008, revised 11 December 2008 and accepted 16 December 2008
- Abstract
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Keywords:
- detection;
- food;
- PCR;
- rapid techniques;
- toxins
Abstract
Aims: To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism.
Methods and Results: Real-time PCR assays based on the detection of the genes ntnh encoding the nontoxin-nonhaemagglutinin (NTNH) proteins or the most homologous regions of the botulinum neurotoxin (bont) genes have been developed together with four real-time PCR assays, each being specific of the genes bont/A, bont/B, bont/E, bont/F and enables a toxin type-specific identification. The specificity of the assays was demonstrated using a panel of botulinum toxin producing clostridia (29 strains), nonbotulinum toxin producing clostridia (21 strains) and various other bacterial strains. The toxin type-specific assays had a sensitivity of 100 fg–1000 fg of total DNA in the PCR tube (25–250 genome equivalents) which correspond to 103 to 104 cells ml−1. After a 48 h enrichment in anaerobic conditions, these PCR assays allowed the detection of Clostridium botulinum type A in a naturally contaminated sample of ‘foie gras’ suspected in a C. botulinum outbreak.
Conclusion: These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism.
Significance and Impact of the Study: Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.