Differentiation of Clostridium perfringens and Clostridium botulinum from non-toxigenic clostridia, isolated from prepared and frozen foods by PCR-DAN based methods

During the elaboration process of prepared and frozen foods, Clostridium sp . have been reported. From those microorganisms, C. perfringens and C. botu linum may pose a high risk for the consumers. To avoid these pathogenic org anisms an HACCP program should be implemented, but in addition sensitive an d moderately time consuming microbiological methods for monitoring C. perfr ingens and C. boulinum should be established. In this work, an RFLP analysi s of the 16S rDNA will be developed to differentiate C. perfringens from ot her Clostridium sp. In addition, a PCR protocol, will be assayed to detect C. botulinum. Both two methods will be compared with biochemical characteri zation by API system. The restriction analysis of the 16S rDNA with Tag I a nd Rsa I showed at least the same sensitivity to differentiate C. perfringe ns from clostridial isolates as biochemical identification. However, the fo rmer method takes only 8-10 h of analysis as compared with 24-48 h required for biochemical characterization. With the specific PCR protocol to detect C. botulinum a band of 1.1 kbp was obtained derived from the specific ampl ification of BoNT genes, taking 6-8 h for analysis. Both two molecular DNA based methods should be considered as verification techniques of pathogenic clostridia in the HACCP program.