Establishment And Preliminary Application Of Multiplex PCR For Toxintyping And An ELISA For Detection Of α-toxin Antibody Of C.perfringens

Clostridium perfringens can cause various diseases. The main pathogenic role of Clostridium perfringens depends on the toxins. According to the ability to product the mainly lethal toxins(α, β, ε and ι). the bacteria will be divided into 5 types: A, B, C, D and E. In order to detecting and typing C.perfringens rapidly and evaluating of α-toxin antibodies after using vaccination, this research established a multiplex PCR method and an ELISA for detection of antibodies, respectively. The main results are as follows:1. Establishment of a multiplex PCR for detecting different types of goat’s C.perfringens strains.Designing four pairs of primers targeting α, β, ε and ι toxins genes and optimizing the reaction condition, the experiment established multiplex PCR for identification and toxintyping of C.perfringens strains. The specificity assay showed that the expected bands of C.perfringens reference strains(A, B, C, D and E) were amplified successfully. However, the bands could not be amplified from Clostidrium novyi(C.novyi), Clostridium septicum(C.septicum) and sterile ddH2 O as negative control groups. The sensitivity assay showed that the limits DNA quantity were 9.0 pg, 17.8 pg, 12.2 pg,13.8 pg and 18.5 pg of five toxin types C.perfringens(A, B, C, D and E), respectively. The repetitive assay had well repeatability. 9type A strains of and 1 type C strains of C.perfringens were detected by the established multiplex PCR from 21 clinical samples of dead goats. This experiment established the multiple PCR method which can detect C.perfringens rapidly and identify five toxin types of C.perfringens.2. Prokaryotic expression, purification and renaturation of C.perfringens α-toxin.According to the published sequence of mature peptides gene of C.perfringens α-toxin,the experiment designed one pairs of primers. Amplifying by PCR and cloning into pET-28 a vector, the prokaryotic expression recombinant plasmid pET-28a-α containing α-toxin gene was successfully constructed. The BL21(DE3) contain recombinant plasmid pET-28a-α was induced by IPTG. Different concebtration of IPTG had little influence on the expression ofα-toxin fusion protein, and the best induction time was 7 h. The expressed form was inclution body. After purified and refolded, western blot showed that the α-toxin fusion protein had well reactiongenicity.3. Establishment of an indirect ELISA for detection of serum antibody of C.perfringensα-toxin.The best indirect ELISA reaction conditions were optimized by using the refoldedα-toxin as coating antigen. The optimal conditions of ELISA were: the coating concentration of α-toxin fusion protein was 5.0 μg/mL, the serum was diluted into 1∶50, the enzyme labeled antibody was diluted into 1∶5 000, sealing time was 1 h, reaction time of antigen and antibody was 30 min. The cut-off value of the assay was 0.393. The specificity was 96.5%and the sensitivity was 98%. The intra-assay C.V were 2%~3% and the inter-assay C.V were less than 8%. A total of 521 sera samples were detected by the indirect ELISA and the positive rate was 92.7%. The establishment and preliminary application of an indirect ELISA makes a solid foundation for detection of antibody against C.perfringens α-toxin antigen.