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 102 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 103 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 CT 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.

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