| Clostridium perfringens is a gram-positive, endospore-forming, anaerobic that has long been recognized as a significant cause of both histoxic and gastrointestinal (GI) disease in human and domestic animals. This bacterium was wildly distributed in soil, water resource, human and animal GI tract. C.perfringens isolates are commonly classified in five types (type A to E), depending upon their ability to express four lethal toxins (alpha, beta, epsilon, and iota toxin), these toxins are secreted into the medium during the exponential growth phase and kill mice when culture supernatant is injected intraperitonealy. The classical typing method is based on mouse lethality test and sero-protection with neutralizing antibodies raised against crude culture supernatants of each representative C.perfringens toxin-type. The serum neutralizing test for typing of C.perfringens are time-consuming and complex-handle. In previous study, a multiplex PCR developed by Qiyi Wen et al was used to identify five types of C.perfringens based on amplifying the genes for the major's four toxins of C.perfringens. The four pairs of PCR primers were designed according to these toxin sequences in genebank and the concentration of each primer was as titrate in a PCR assay to allow concurrent amplification of multiplex target sequences and other parameters of this assay were optimized. Other two pairs of primers were also designed in this PCR toxin-genotype assay based on the two important toxins - enterotoxin ( CPE) and B2- toxin. Comparing genotype with phenotype in vivo test neutralization carried out specificity of the assay. Six multiplex PCR products were amplified and obtained after multiplex PCR reaction from C.perfringens isolates, these included fragments of cpa(324bp), cpb(196bp), ext(655bp), iA(446bp), cpe(233bp) and cpb2(567bp). In this study, 346 fecal samples and 275 food samples were collected, then isolate C.perfringens from these samples with conventional method. These isolates were identified bybiochemical test and the multiplex PCR reaction.69 C.perfringens isolates were isolated from 346 fecal samples in this study. C.perfringens isolates are accounted for 19.9% of total food samples including human feces, pig feces, chickens feces, and all C.perfringens isolates were proved from samples by the multiplex PCR assay. Our results showed that all of C.perfringens isolates was be found to be type A and 47.8% C.perfringens carrying fa toxin was present in this samples. Enterotoxigenic C.perfringens isolates and other types C.perfringens were not found in this investigation.A total of 97 C.perfringens isolates was isolated from 275 retail food samples in this study, C.perfringens isolates are accounted for 35.3% of total food samples including porks, chickens, sea foods. All isolates were identified to be type A C.perfringens by multiplex PCR genotyping assay, other types C.perfringens isolates was not identified from food samples, 20.6% of type A C.perfringens isolates being carried B2 toxin was in all food isolates and 2 cpe-positive isolates were identified from 275 food samples, -2.1% from total food samples in this investigation.About 5% of C.perfringens type A isolates carry the gene encoding the CPE, C.perfringens type A isolates producing CPE are an important cause food poisoning and non-foodborne human gastrointestinal(GI) disease, including antibiotic associated diarrhea(AAD) and sporadic diarrhea. Recent studies suggest that C.perfringens type A food poisoning is caused by C.perfringens isolates carrying a chromosomal cpe gene, while CPE-associated with non foodborne human GI disease, such as AAD or sporadic diarrhea, are caused by cpe-IS1151 or cpe-IS1470-like plasmid cpe C.perfringens isolates.The 960bp cpe ORF gene in one cpe C.perfringens isolate was amplified by single PCR reaction. Then this 960bp PCR product was sequenced and was the same as the cpe ORF sequence in Genbank.The new multiplex PCR was developed that can rapidly distinguish cpe-genotype of enterotoxigenic type A isolates (i.e., determine-wh...
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