Establishment Of Multiplex PCR Assay For BVDV,Pm And Kp In Dairy Cows

In recent years,the pathogens of the bovine respiratory disease(BRD)have gradually expanded along with the development of dairy farming in China,and the types of mixed infections between pathogens have increased.This phenomenon is not only the diagnosis and treatment become more difficult of diseases.It is also a huge challenge for dairy farming and the economy.Pathogenic microorganisms causing the onset of cattle,such as Pasteurella multocida(Pm),Klebsiella pneumoniae(Kp),bovine Viral Diarrhea-mucosal virus(BDVD)will can induce BRD,and even lead to cow death,seriously endangering The development of the cattle industry.so,the strengthening of research on this disease are extremely important.In order To establish a multiplex PCR assay for the important pathogens Pm,Kp and BVDV of respiratory diseases in the dairy cow population in Hebei Province to study.This study collected the respiratory disease diseases of large-scale dairy farms in Hebei Province as the research object.RT-PCR detection and MDBK cell proliferation method were used to separate BVDV.Traditional bacterial isolation and 16 SrRNA sequence analysis were used to isolate and identify pathogenic bacteria.At the same time,the pathogenicity tests of Kp and Pm isolated in this experiment were carried out in mice,and the drug resistance of the isolates was analyzed by drug sensitivity test.According to the specific conserved sequences of Kp,Pm and BVDV published in Genbank,namely kmt gene,Phe0 gene and E0 gene,Primer 5 software was used to design and synthesize specific primers to establish a multiplex PCR reaction system.The final concentration of the primer,the optimal template ratio,the optimal annealing temperature,the optimal annealing time and the number of cycles were optimized and screened.The specificity,sensitivity and repeatability of the optimized reaction system were verified and the performance of the method was evaluated.Finally,34 cases of clinical respiratory diseases were detected simultaneously by the established multiplex PCR method and traditional single pathogen detection method,which provided data support for the clinical application of the method.In this experiment,one strain of bovine viral diarrhea-mucosal disease virus,four strains of Klebsiella pneumoniae and three strains of Pasteurella multocida type A were isolated from the disease material;the amplified fragment of the E0 gene sequence of BVDV isolate was obtained The homology of BVDV reference strain distributed with NCBI was 99.8%,the homology of the phoE gene sequence of Kp isolate was Above 96.6%,and the homology of the kmt gene sequence of the Pm isolate was Above 95.5%;all the mice inoculated with Kp and Pm isolates died and had strong pathogenicity;Kp isolates were moderately sensitive to three antibiotics such as ofloxacin,amikacin and neomycin,Pm separation The strain is sensitive to four antibiotics,namely ciprofloxacin,ofloxacin,florfenicol and amikacin,and exhibits different degrees of resistance to other test drugs.A multiplex PCR assay for bovine viral diarrhea-mucosal disease virus,Klebsiella pneumoniae and Pasteurella multocida was established.The optimal template ratio was 1?L,1.3?L and 0.7?L,and the final primer concentration was 0.2?mol/L,1.25?mol/L and 0.5?mol/L,the optimum annealing temperature is 58.1°C,the optimum annealing extension time is 40 s,and the optimal cycle number is 35 times.The multiplex PCR assay established in this experiment has good reproducibility and specificity results,and The sensitivity of pathogens Pm and Kp both are 10fg/?L,and the sensitivity of BVDV is 100fg/?L.The newly established multiplex PCR detection method is consistent with the clinical detection results of the traditional bacterial isolation and identification method and the virus single PCR detection method,and can achieve the bovine viral diarrhea-mucosal disease virus,Pasteurella multocida and Klebsiella pneumoniae three kinds of pathogens Rapid and accurate identification.In this study,we used the isolated pathogens BVDV,Kp and Pm to establish a multiplex PCR assay with high specificity and high sensitivity.In clinical application,the detection conformity of the method reached 100%,achieving fast and accurate Identification results,can be applied.