Advertisement

Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum

Authors

  • P. Fach,

    1.  Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d’Etudes et de Recherches sur la Qualité des Aliments et les Procédés Agro-alimentaires (LERQAP), Maisons-Alfort, France
    Search for more papers by this author
  • P. Micheau,

    1.  Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d’Etudes et de Recherches sur la Qualité des Aliments et les Procédés Agro-alimentaires (LERQAP), Maisons-Alfort, France
    Search for more papers by this author
  • C. Mazuet,

    1.  Institut Pasteur, Centre de référence des bactéries anaérobies, Paris, France
    Search for more papers by this author
  • S. Perelle,

    1.  Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d’Etudes et de Recherches sur la Qualité des Aliments et les Procédés Agro-alimentaires (LERQAP), Maisons-Alfort, France
    Search for more papers by this author
  • M. Popoff

    1.  Institut Pasteur, Centre de référence des bactéries anaérobies, Paris, France
    Search for more papers by this author

Patrick Fach, Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Laboratoire d’Etudes et de Recherches sur la Qualité des Aliments et les Procédés Agro-alimentaires (LERQAP), 23 avenue du général De Gaulle, 94700 Maisons-Alfort, France.
E-mail: p.fach@afssa.fr

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.

Ancillary