Development of an in vitro method for detection of Clostridium botulinum types A and E using real-time PCR

Abstract

Specific primers for C. botulinum types A and E neurotoxin genes were evaluated both from the literature and of own construction. Furthermore, a real-time system with specific hybridisation probes was designed for C. botulinum type E neurotoxin gene, and is under construction for type A. Sequencing of part of the neurotoxin gene for type E showed some differences with sequences reported in GenBank. The PCR methods were optimised regarding amplification efficiency, linear range and specificity. The detection limit for type E using real-time PCR is at least 0,1 ng/ml, corresponding to 0,4 pg of total DNA in the tube, and at least 0,5 ng/ml (2,5 pg of total DNA) for type A using conventional PCR. Quantitative reverse transcription PCR was used to study the relative expression of the neurotoxin gene in different growth phases.