Establishment And Application Of Quadruple Fluorescence PCR Detection Method For Bovine Respiratory Virus Disease

The main causative factor of bovine respiratory disease is Bovine Herpes Virus1(BHV-1),Bovine Respiratory Syncytial Virus(BRSV),Bovine Parainfluenza Virus Type 3(BPIV-3)and Bovine Viral Diarrhea Virus(BVDV).Specific primers and Taq Man fluorescent probes were designed according to the conserved region sequences of the gB gene of BHV-1,the F gene of BRSV,the M gene of BPIV-3 and the 5’UTR gene of BVDV.After optimization of conditions,the multiplex fluorescence PCR method was verified by specificity test,sensitivity test and repeatability test and the establishment of a standard curve,and the quadruple fluorescence PCR detection method of BHV-1,BRSV,BPIV-3 and BVDV was successfully established.The results show that the method has no specific amplification for Brucella bovis,swine fever virus,petitella ruminant virus,Pasteurella multocida,and BHV-1,BPIV-3 and BVDV can detect as low as8.268 copies/μL,BRSV can detect as low as 82.68 copies/μL.The CV value ranges from 1 %to 2 %,which proves that the method has strong specificity,high sensitivity,good repeatability and stability.This method was used to detect 1489 samples from different slaughterhouses in Hunan Province.The results showed that 4 pathogens,including BHV-1,BRSV,BPIV-3 and BVDV,were detected.The positive rates were 0.74 %,1.07 %,0.67 % and 1.07 % respectively,in which there is a mixed infection of viruses.This study established a multiplex fluorescent PCR method that can rapidly detect BHV-1,BRSV,BPIV-3 and BVDV at the same time,which provides a fast and simple method for the differential diagnosis and detection of bovine respiratory virus diseases.