Abstract
The polymerase chain reaction (PCR)-based detection methods of Clostridium perfringens enterotoxin (CPE) gene (cpe) from food and environmental samples and DNA fingerprinting methods for molecular epidemiologic analysis, i.e., restriction fragment length polymorphism analysis of PCR-amplified cpe regions (PCR-RFLP) and random amplified polymorphic DNA (RAPD), were investigated. We newly designed four primer regions within a cpe gene, and then developed two PCR amplification systems, CP-5' and CP-89. Both systems generated the expected sizes of amplicons, 957-bp and 207-bp, respectively from CPE-positive C.perfringensstrains. A nested PCR method, consisting of the first PCR by the CP-1 system, which we previously established, followed by a second PCR by the CP-89 system, could detect differences at a 100 level per reaction tube. To evaluate the usefulness of these PCR systems, we applied those to foods in a C.perfringensoutbreak, from which CPE-positive C.perfringenswas isolated by an enrichment culture procedure, and environmental samples such as those in soil and river sediment. The nested PCR method detected cpe genes in three of the four tested foods without enrichment. The CP-89 system combined with enrichment with TGC medium (TGC-PCR) detected cpe genes from 37 (77%) of 48 environmental samples, suggesting a higher level of distribution of this pathogen than that previously considered . For PCR-RFLP analysis of the amplicons by the CP-5' system and DNA fingerprinting by RAPD analysis, PCR-RFLP analysis showed an identical pattern except for a strain. RAPD patterns generated showed identical patterns within each outbreak and different patterns between outbreaks. These results indicated that the PCR methods and the molecular epidemiologic methods examined here may be available for the investigation of enterotoxigenic C.perfringens.
