Establishment And Application Of Double Fluorescence Quantitative PCR Method For Detection Of Clostridium Perfringens And Pasteurella Multocida In Bovis

Clostridium percapsulens and Pasteurella multocida can both cause sudden death in cattle and cause huge losses to the cattle industry.Because these two pathogens are often recessive or mixed infection,and the onset is rapid,and the clinical symptoms are similar,it is difficult to make clinical diagnosis.With the development of molecular biology,especially the rapid popularization of fluorescence quantitative PCR detection technology,some scholars have established fluorescence quantitative PCR detection methods,but the double fluorescence quantitative PCR detection methods of Pas-teurella multocida and Clostridium perfringens are rarely reported.Therefore,it is of great significance for the prevention and control of bovine disease to establish a double fluorescence quantitative PCR method for detection of Pasteurella multocida and Clos-tridium percapsulens in the laboratory for rapid and efficient differential diagnosis.In order to establish a dual fluorescence quantitative PCR assay with strong spec-ificity,high sensitivity and good repeatability,this study designed and synthesized spe-cific primers and probes according to the conserved gene sequences of Pasteurella mul-tocida and Clostridium perfringens,explored the optimal reaction conditions,and ver-ified the sensitivity,specificity,repeatability and clinical samples.The results show that:The method showed good sensitivity,specificity and reproducibility.The detection lim-its of Pasteurella multocida and Clostridium perfringens were 10~1 copies/μL,and had a good linear relationship in the range of 10~3 copies/μL to 10~8 copies/μL.At the same time,it did not cross-react with Streptococcus agalactiae,Streptococcus mammary,Streptococcus agalactiae,Staphylococcus aureus,listeria monocytogenes,Enterococ-cus faecalis,Klebsiella,Escherichia coli,Salmonella,Brucella,Mycobacterium tuber-culosis,Mycobacterium para-tuberculosis and other common pathogens of cattle.The coefficient of variation between groups and within groups was less than 4%.It can be used for the rapid differential diagnosis of Pasteurella multocida and Clostridium perfringens.Using this method,14 strains of Pasteurella multocida and 16 strains of Clostridium perfringens were successfully isolated from positive dead cattle tissue sam-ples.Further PCR analysis showed that 9 strains of Pasteurella multocida were type A and 5 were type B.Seven strains of Clostridium perfringens were type A and nine were type C.In conclusion,the dual fluorescence quantitative PCR method for detection of Pas-teurella multocida and Clostridium perfringens established in this study has high sensi-tivity,specificity and stability,and can be widely used in laboratory differential diag-nosis,providing technical support for the control of Pasteurella multocida and Clostrid-ium perfringens.