Zhuandı GONG, Xıaoyun SHEN, Haoqın LIANG, Jınjıng GENG, Suocheng WEI

TaqMan probe qRT-PCR detects bovine parvovirus and applies clinically

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 of conserved 5?-untranslated regions 5'-UTR of strain Haden of BPV. Optimum reaction conditions were comparatively selected. The sensitivity, specificity, and reproducibility of TaqMan probe qRT-PCR were measured respectively. Feces specimens collected from diarrhea 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 detect BPV, but not for other viruses JEV, CSF, RABV, BRV, BVDV, and bFMDV . The detection limit was 5.26 copies/μL. Both intraassay and interassay VCs were lower than 1.65%. Detection of 308 feces samples showed 36 positives for TaqMan probe qRT-PCR compared to 32 positives of universal PCR. Coincidence rate of the two methods was 88.89%. In conclusion, the established TaqMan probe qRT-PCR could specifically detect BPV. It had excellent sensitivity, specificity, and stability. The detection limit was 5.26 copies/μL. Its sensitivity was 10,000-fold over the conventional PCR. TaqMan probe qRT-PCR will help to enhance diagnosis and therapy efficacy of diarrhea calves.

Keywords:

Bovine parvovirus, TaqMan probe, real-time PCR, sensitivity, specificity,

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