Investigation Of Clostridium Perfringens Toxin Types Isolated From Animals In Shandong Province By Multiplex-PCR

In order to confirm the real Epidemical situation of Clostridium perfringens toxin types in Shandong Province, multiplex-PCR was established, by which we investigated 692 samples isolated from Dezhou,Taian, Zaozhuang,Qingdao,Linyi and Yantai. We detect 124 Clostridium perfringens positive,and all are type A.The survey result is absolutely true,which provide theory guide for previntion. The study established a fast PCR detect method to Clostridium perfringens,which has high specifity and possesses latent application prospect.The study consists of four parts.Part Ⅰ:Isolation and Toxin-type Identification of Clostridium perfringens of samples. 124 strains of Clostridium perfringens were isolated from animals feces. The study result showed isolation strains were toxin type A by API-20A biochemical test method.Part Ⅱ:A pair of primer was succeeded in designing and synthesizing according to the published sequence of the α -toxin of Clostridium perfringens.The PCR can amplify 325bp specific α -toxin fragments.It accords with the predicted amplification segament, however,it has no positive result with system to amplify Salmonella , E-coli , Enterococcus and Staphylococcus,so ,it can demonstrate the PCR amplification system has good specificity.In addition,it also possesses very high sensitivity because the system can detect the 1CFU/ml dilute template. The PCR technique was applied tor the detection of Clostridium perfringens in localized areas of Shandong province.The result showed that 21 of 102 samples were positive which is basically identical with other bacteria isolation methods.According to the above results ,the study has developed a Clostridium perfringens quick detection method which possesses latent application prospect. Part Ⅲ:For detection of Clostridium perfringens toxin types four pairs of primer for alpha, beta, epsilon and jota toxin gene were succeeded in designing and synthesizing according to the published sequence.It candistinguish type A,B,C,D and E of Clostridium perfringens.The specific of PCR is high,only Clostridium perfringens show positive,other strains such as Salmonella > E-coli^ Enterococcus and Staphylococcus all show negative. The multiplex PCR was applied for the identification of toxin types of Clostridium perfringens from samples which were collected from cattle stable and had been identified by ELISA.The PCR result accord with the ELISA method.So the muliplex-PCR can be applied to epidemiology survey. Part IV: The multiplex PCR was applied for the identification of toxin types of Clostridium perfringens from samples which were collected from earlier epidemic districts of Shandong province. The results showed that all isolated 124 samples (17.9%) were Clostridium perfringens toxin type A, that was alpha toxin positive which is different from abroad reports.