Establishment Of Multiplex PCR Method For Detection Of Clostridium Perfringens And Expression Of Phage Lyase In Eukaryotic System

Clostridium perfringens is a bursting,sudden death disease that has a serious impact on the farming industry and is prevalent throughout the world.The traditional laboratory test method is time-consuming and cannot be typed,which has limitations for the diagnosis and prevention of the disease.Therefore,it is extremely important to establish an efficient,accurate and rapid detection method.With the complete ban of antibiotics as additives in feeds,new challenges are posed for the farming industry in the prevention and treatment of bacterial diseases.As a highly specific and efficient bacterial lysis protein,phage lysozyme has re-emerged in the "post-antibiotic era".Based on the gene sequences of Clostridium perfringens α,β,ε and ι toxins published in GenBank,four pairs of primers were designed and synthesized to target the four toxins.And the multiplex PCR method was optimized by changing the reaction conditions such as primer concentration and annealing temperature.The results showed that the α,β,ε and ι toxin genes were amplified with the expected size of the target bands,while the bands of S.aureus,Pasteurella multocida,S.dysgalactiae equisimilis,Klebsiella pneumoniae and E.coli did not appear;when the nucleic acid concentration was reduced to 12.5 ng/mL,the bands corresponding to the α,β,ε and ιtoxin genes could still be clearly amplified.When the nucleic acid concentration was reduced to 3.12 ng/mL,the amplified bands of a and εtoxin genes could still be observed.The method was applied to 68 nasal swabs from cattle suspected of sudden death caused by C.perfringens from 17 farms in Ningxia,of which 52 samples tested positive for C.perfringens type C and the remaining 16 samples tested negative,representing a positive rate of 76%.The Clostridium perfringens phage lytic enzyme CP3626 gene was synthesized artificially and optimized according to the codon preference of the B.bicolor expression system.The PPICZα-3626 expression vector was constructed,electrotransferred in.