Objective A multiplex RT-PCR assay for rapid and simultaneous detection of akabane virus (AKAV), bovine herpesvirus 1 (BoHV-1), and bovine viral diarrhea virus (BVDV) was developed.

Method Three pairs of primers were designed for the conservative regions of S genes of AKAV, gH of BoHV-1, and 3'UTR of BVDV for establishing an RT-PCR assay capable of detecting the three viruses simultaneously under optimized reaction conditions.

Result  The assay detected positive only on AKAV, BoHV-1, and BVDV but negative on bovine parvovirus (BPV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus 3 (BPIV-3), foot-and-mouth disease virus (FMDV), and bovine ephemeral fever virus (BEFV). The minimum detection thresholds of the assay were 1.0×10³ copies·μL⁻¹ on the recombinant plasmids of the three target viruses with high intra- and inter-batch reproducibility. On 143 whole blood/tissue specimens of cattle with reproductive disorders that included cases with mixed infections, it delivered the positivity rates on AKAV, BoHV-1, and BVDV of 2.80% (4/143), 21.68% (31/143), and 38.46% (55/143) as well as the conformity rates with the local standards for AKAV, BoHV-1, and BVDV of 99.3%, 98.6%, and 97.2% with the Kappa coefficients of 0.885, 0.960, and 0.942 respectively.

Conclusion The newly established multiplex RT-PCR assay displayed high specificity, sensitivity, and reproducibility in detecting AKAV, BoHV-1, and BVDV simultaneously with a considerably reduced operation cost.