Isolation And Identification Of Clostridium Perfringens And Establishment Of PCR Detection Method

Clostridium perfringens is a opportunistic pathogen causing a wide range of zoonotic diseases,in order to understand the distribution of Clostridium perfringens type in cattle and sheep in Inner Mongolia,4 pairs of primers were designed according to the GenBank published the 16S rRNA gene of Clostridium perfringens and Genes encoding alpha,beta,epsilon toxin.Then,established of PCR method for detection of Clostridium perfringens 16S rRNA and multiplex PCR typing method for Clostridium perfringens alpha,beta and epsilon toxin.Meanwhile,detected the bacteria isolated,cultured and identified in 64 samples collected.The recombinant plasmid was constructed by 16S rRNA,which was identified as PCR positive bacteria isolated from Clostridium perfringens and analyzed the homology with the reference strains of Clostridium perfringens and constructed the phylogenetic tree.The test results showed that establishment of Clostridium perfringens 16S rRNA PCR method and multiplex PCR typing method for Clostridium perfringens alpha,beta and epsilon toxin was amplified corresponding bands,the sensitivity and specificity of PCR were relatively high.In this study,the bacteria which were isolated and cultured from 64 samples collected and identified by gram staining had 11 strains which were consistent with Clostridium perfringens.The two PCR methods were used for the detection of these 11 isolated bacteria.The amplified bands were consistent with the expected results.Multiple PCR results are shown that 3 strains of Clostridium perfringens type A,3 strains of Clostridium perfringens type C and 5 strains of Clostridium perfringens type Dwere isolated from 11 strains isolated bacteria.Sequencing results showed 11 strains Clostridium perfringens of 16S rRNA are 98%similarity with reference strain of 16S rRNA.