Establishment And Application Of Quadruple Quantitative PCR Method For Detection Of Major Bacterial Pathogens In Bovine Respiratory Tract

Bovine respiratory disease syndrome is a general term for bovine pneumonia,transport fever,bronchitis and other diseases caused by a variety of viruses or bacteria.It is a serious problem prevalent in cattle farms around the world and brings huge economic losses to the breeding industry.In order to quickly and easily detect the main bacterial pathogens of bovine respiratory diseases,two sets of primers and probes were designed for the KMT gene of P.multocida and the Omp A gene of H.somni,respectively.For M.haemolytica and M.bovis,the primer and probe sequences of t RNA gene and OPPO gene in the literature were cited,and the annealing temperature and the amount of primer and probe were optimized.Finally,a quadruple fluorescence quantitative PCR method for simultaneous detection of P.multocida,M.haemolytica,M.bovis and H.somnus was established.The results showed that the quadruple fluorescence quantitative PCR method established in this experiment had specific amplification curves for P.multocida,M.haemolytica,M.bovis and H.somnus,and had no cross-reaction with other pathogens,indicating that the specificity was good.The minimum detection limits of P.multocida,M.haemolytica,M.bovis and H.somni genomic DNA were 50copies/μL,50copies/μL,50copies/μL and 10copies/μL,respectively,indicating high sensitivity.The correlation coefficients R~2of the standard curve were 0.9957,0.994,0.994 and 0.9938,respectively,showing good linear characteristics.The intra-batch and inter-batch coefficients of variation were less than 3%,indicating good repeatability.Compared with WOAH and industry standard PCR methods,the coincidence rate of the established quadruple fluorescence quantitative PCR method was 93.3%for P.multocida,95.6%for M.haemolytica and 97.8%for M.bovis.The method was used to detect 396 clinical nucleic acid samples.The infection rates of P.multocida,M.haemolytica,M.bovis and H.somni were 29%,17%,2%and 14%,respectively.The mixed infection rates of P.multocida and M.haemolytica,P.multocida and M.bovis,P.multocida and H.somni were 4.54%,5.00%and 3.28%,respectively.In this study,a sensitive,specific,multiplex and rapid quadruple fluorescence quantitative PCR detection method for bacterial pathogens of bovine respiratory tract was successfully established,which provided a reliable basis for the diagnosis,prevention and control of bovine respiratory tract diseases.