The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic proto- col which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5′ - and 3′-untranslated regions (5’UTR, 3’UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were tran- scribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excel- lent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms.
Sapelovirus A (SV-A) is a positive-sense single-stranded RNA virus which is associated with acute diarrhea, pneumonia and reproductive disorders. The virus capsid is composed of four proteins, and the functions of the structural proteins are unclear. In this study, we expressed SV-A structural protein VP1 and studied its antigenicity and immunogenicity. SDS-PAGE analysis revealed that the target gene was expressed at high levels at 0.6 mM concentration of IPTG for 24 h. The mouse polyclonal antibody against SV-A VP1 protein was produced and reached a high antiserum titer (1: 2,048,000). Immunized mice sera with the recombinant SV-A VP1 protein showed specific recognition of purified VP1 protein by western blot assay and could recognize native SV-A VP1 protein in PK-15 cells infected with SV-A by indirect immunofluorescence assay. The successfully purified recombinant protein was able to preserve its antigenic determinants and the generated mouse anti-SV-A VP1 antibodies could recognize native SV-A, which may have the potential to be used to detect SV-A infection in pigs.