Establishment And Application Of Quadruple Fluorescence Quantitative PCR Method For IBRV,BVDV,BPIV3 And BRSV

Bovine respiratory diseases have caused great economic losses to the cattle industry.It is difficult to distinguish single infection or mixed infection clinically,and it is impossible to formulate precise prevention and control measures.The establishment of quadruple fluorescent quantitative PCR detection methods for infectious bovine rhinotracheitis virus(IBRV),bovine viral diarrhea virus(BVDV),bovine parainfluenza virus type 3(BPIV3)and bovine respiratory syncytial virus(BRSV)is conducive to early clinical diagnosis and differential diagnosis.In this study,a quadruple fluorescence quantitative PCR method was established using IBRV g B gene,BVDV 5’UTR gene,BPIV3 P gene and BRSV N gene.The standard curve correlation coefficients R~2of IBRV,BVDV,BPIV3 and BRSV were1.000,1.000,0.995 and 0.997,respectively,and the amplification efficiency E was98.5%,102.0%,108.1%and 103.1%,respectively,showing good linear characteristics.This method had no cross reaction with Mycoplasma,Mannheimia haemolytica,Bovine rotavirus,Klebsiella pneumoniae,Pasteurella multocida type A,and had good specificity.The minimum detection limit of IBRV,BVDV and BRSV was 50 copies/μL,and BPIV3 was 10 copies/μL.The intra-batch and inter-batch coefficients of variation were≤1.2%,and the repeatability was good.The coincidence rate of the established quadruple fluorescence quantitative PCR method and the RT-q PCR method of WOAH,national standard and patent was IBRV 92.47%,BVDV 91.39%,BPIV3 87.09%and BRSV 97.84%.The positive rates of IBRV,BVDV,BPIV3 and BRSV were 7.25%,12.63%,10.48%and 10.38%,respectively.In this study,a rapid,sensitive and specific quadruple real-time fluorescence quantitative PCR method was established to provide technical support for the differential diagnosis and early diagnosis of bovine respiratory diseases.