Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producingClostridium botulinum,Clostridium baratiiandClostridium butyricum

Abstract

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 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. 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.