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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
Author(s) -
Fach P.,
Micheau P.,
Mazuet C.,
Perelle S.,
Popoff M.
Publication year - 2009
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2009.04215.x
Subject(s) - clostridium botulinum , clostridium butyricum , clostridiaceae , clostridiales , microbiology and biotechnology , botulism , clostridium , biology , bacteria , toxin , genetics
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 10 3 to 10 4 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.