Triplex qRT-PCR with specific probe for synchronously detecting Bovine parvovirus, bovine coronavirus, bovine parainfluenza virus and its applications.

Bovine parvovirus (BPV), bovine coronavirus (BCoV) and bovine parainfluenza virus (BPIV) are common etiologies causing gastrointestinal and respiratory diseases in dairy herds. However, there are few reports on the synchronous detection of BPV, BCoV and BPIV. The present article aimed to develop a quick and accurate RT-PCR assay to synchronously detect BPV, BCoV and BPIV based on their specific probes. One pair universal primers, one pair specific primers and one specific probe was designed and synthesized. After the concentrations of primer and probe and annealing temperature were strictly optimized, the specificity, sensitivity and repeatability of the established triplex probe qRT-PCR were evaluated, respectively. The results showed the recombinant plasmids of pMD18-T-BPV, pMD18-T-BCoV and pMD18-T-BPIV were 554bp, 699bp and 704bp, respectively. The optimal annealing temperature was set at 45.0°C for triplex qRT-PCR. The triplex probe qRT-PCR can only synchronously detect BPV, BCoV and BPIV. Detection sensitivities were 2.0×102, 2.0×102 and 2.0×101 copies/μL for BPV, BCoV and BPIV, being 1000-fold greater than that in the conventional PCR. Detection of clinical samples demonstrated that triplex probe qRT-PCR had a higher sensitivity and specificity. The intra-assay and inter-assay coefficient of variation were lower than 2.0%. Clinical specimens verified that the triplex qRT-PCR had a higher sensitivity and specificity than universal PCR. In conclusion, this triplex probe qRT-PCR could detect only BPV, BCoV and BPIV. Minimum detection limits were 2.0×102 copies/μL for BPV and BCoV, and 2.0×101 copies/μL for BPIV. The sensitivity of this triplex probe qRT-PCR was 1000-fold greater than that in the conventional PCR. The newly qRT-PCR could be used to monitor or differentially diagnose virus infection.