Objective  To establish a multiplex RT-PCR method for the rapid and simultaneous detection of akabane virus （AKAV）, Bovine herpesvirus 1 （BoHV-1） and bovine viral diarrhea virus （BVDV）, so as to lay a foundation for the differential diagnosis, prevention and control of bovine reproductive disorder epidemics.

  Method  Three pairs of primers were designed for the conservative regions of AKAV S gene, BoHV-1 gH gene and BVDV 3'UTR gene, and a multiplex RT-PCR assay for AKAV, BoHV-1 and BVDV was established through the optimal of reaction condition parameters.

  Result  The specificity results showed that the method was positive only for AKAV, BoHV-1 and BVDV, and negative for the nucleic acids of bovine parvovirus （BPV）, bovine respiratory syncytial virus （BRSV）, bovine parainfluenza virus 3 （BPIV3）, foot-and-mouth disease virus （FMDV）, and bovine ephemeral fever virus （BEFV）. The sensitivity results showed that the lower threshold of this method for AKAV, BoHV-1 and BVDV recombinant plasmids were all 1.0×10³ copies/μL. The results of the reproducibility test showed good intra- and inter-batch reproducibility. The established method was used to determine 143 whole blood/tissue samples of cattle with reproductive disorders and compared with the existing local standards to verify the efficacy of this test for practical clinical application. The results showed that the positivity rates of AKAV, BoHV-1 and BVDV were 2.80% （4/143）, 21.68% （31/143） and 38.46% （55/143）, respectively, with the presence of several mixed infections; and the conformity rates of this method with the local standards for AKAV, BoHV-1 and BVDV were 99.3%, 98.6% and 97.2%, respectively. The Kappa coefficients of the two were 0.885, 0.960 and 0.942 respectively, indicating that the method established in this study was in good agreement with the local standards.

  Conclusion  In this study, we pioneered a multiplex RT-PCR assay with high specificity, sensitivity and low cost for the differential detection of AKAV, BoHV-1 and BVDV.