Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food

The TaqMan real-time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence-specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10-fold dilution series of DNA and spores. The DNA and spores were detected up to level of 0.1 ng/ml and 10(2) spores/ml, respectively. Spore spiked food sample preparation prior to the real-time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 102 spores/ml for spiked sausage slurry, and 10(3) spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 102 spores/ml in both sausage and canned corn. Therefore the real-time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical C, values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real-time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.