ObjectiveTo establish real-time PCR assays for the toxin gene detections of Clostridium botulinum type A and B, construct standard curves and evaluate the specificities, sensitivities and detection thresholds of the assays, and provide evidence for the rapid and accurate detection of C. botulinum.

MethodsSpecific primers and probes were designed based on the sequences of toxin genes of C. botulinum type A and B. Real-time PCR assays were established with optimized reaction conditions. 25 other intestinal bacteria and common bacteria were used to test the specificities of the assays. Standard curve construction, fecal sample simulation and sensitivity measurement were achieved with recombinant plasmids containing toxin genes of C. botulinum type A and B.

ResultsThe specificities of the real-time PCR assays were high. Specific amplification curves were observed in recombinant plasmids containing toxin genes of C. botulinum type A and B. No specific amplifications were found for the 25 other bacteria. The detection thresholds of toxin genes of C. botulinum type A and type B were 5.04×10² copy/μl and 6.91×10² copy/μl respectively according to the amplification curves. The detection thresholds of recombinant plasmids containing toxin genes of C. botulinum type A and B in artificial fecal samples were 1.71×10³ copy/μl and 2.14×10³ copy/μl respectively.

ConclusionIn this study, real -time PCR assays for the toxin gens detections of C. botulinum type A and type B of China were established, which can be applied in the rapid detection of C. botulinum.