TaqMan probe qRT-PCR detects bovine parvovirus and applies clinically

Abstract

The present study aimed to establish a TaqMan probe real-time PCR (qRT-PCR) for detecting Bovine parvovirus (BPV),and to develop a novel diagnostic method of BPV. TaqMan probe and primers were designed and synthesized from the sequences ofconserved 5′-untranslated regions (5′-UTR) of strain Haden of BPV. Optimum reaction conditions were comparatively selected. Thesensitivity, specificity, and reproducibility of TaqMan probe qRT-PCR were measured respectively. Feces specimens collected fromdiarrhea calves were detected using this assay. The results showed that optimized conditions for the probe qRT-PCR were 0.8 μL probes(18.0 μmol/L), 0.4 μL primers (10.0 μmol/L) and annealing temperature of 43.2 °C for 40 s. The probe qRT-PCR could specially detectBPV, but not for other viruses (JEV, CSF, RABV, BRV, BVDV, and bFMDV). The detection limit was 5.26 copies/μL. Both intraassay andinterassay VCs were lower than 1.65%. Detection of 308 feces samples showed 36 positives for TaqMan probe qRT-PCR compared to 32positives of universal PCR. Coincidence rate of the two methods was 88.89%. In conclusion, the established TaqMan probe qRT-PCRcould specifically detect BPV. It had excellent sensitivity, specificity, and stability. The detection limit was 5.26 copies/μL. Its sensitivitywas 10,000-fold over the conventional PCR. TaqMan probe qRT-PCR will help to enhance diagnosis and therapy efficacy of diarrheacalves.