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

• Published in: Journal of applied microbiology Vol. 107; no. 2; pp. 465 - 473
• Main Authors: Fach, P, Micheau, P, Mazuet, C, Perelle, S, Popoff, M
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-08-2009; Blackwell Publishing Ltd; Blackwell; Wiley
• Subjects: Animals; Bacterial Proteins - genetics; Biological and medical sciences; Botulinum Toxins - genetics; Botulinum Toxins - isolation & purification; Clostridium; Clostridium - genetics; Clostridium - isolation & purification; Clostridium botulinum; Clostridium butyricum; detection; DNA Primers - genetics; DNA, Bacterial - analysis; DNA, Bacterial - genetics; Fatty Liver - microbiology; food; foods; Fundamental and applied biological sciences. Psychology; Geese; Hemagglutinins - analysis; Hemagglutinins - genetics; Humans; Life Sciences; Microbiology; Microbiology and Parasitology; Molecular Sequence Data; PCR; Polymerase Chain Reaction - methods; rapid techniques; Sensitivity and Specificity; toxins; Clostridium botulinum; Clostridium butyricum; Enzyme; Applied microbiology; Clostridiales; Metalloendopeptidases; rapid techniques; Foodstuff; Real time; food; Polymerase chain reaction; Peptidases; Toxin; Bontoxilysin; Rapid technique; Analysis method; Clostridiaceae; toxins; Bacteria; Hydrolases; Detection; PCR; detection
• ISSN: 1364-5072; 1365-2672
• DOI: 10.1111/j.1365-2672.2009.04215.x

To develop real-time PCR assays for tracking and tracing clostridia responsible for human botulism. 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. These PCR tests are specific and reliable for detection of heterogeneous BoNT producing clostridia responsible for human botulism. Adoption of these PCR assays is a step forward a reliable and rapid detection of these clostridia in food samples.