TaqMan probe qRT-PCR detects bovine parvovirus and applies clinically

Zhuandi Gong,Suocheng Wei,Haoqin Liang,Xiaoyun Shen,Jinjing Geng

doi:10.3906/vet-1907-80

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 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 &#956L probes 18.0 &#956mol/L , 0.4 &#956L primers 10.0 &#956mol/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/&#956L. 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/&#956L. 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.

Highlights

Bovine parvovirus (BPV) was firstly identified in the 1960s in diarrhea calves [1]
Construction of standard plasmids After the BPV DNA was amplified, the bands of agarose gel electrophoresis showed the PCR product was 554 bp, which was consistent with the expected product sizes (Figure 1)
Optimization of qRT-PCR Based on the screening of the different concentrations of specific primers and probes as well as annealing temperatures, the optimum reaction conditions for TaqMan 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

Summary

Introduction

Bovine parvovirus (BPV) was firstly identified in the 1960s in diarrhea calves [1]. BPV is a member of the genus Bocaparvovirus with a nonenveloped capsid [2]. The viral capsid plays a critical role in the infection of host cells by adhering the virus to specific receptors on the target cells [3]. The genome comprises single-stranded DNA (ssDNA) [4,5] and possesses three open reading frames (ORFs; ORF1, ORF2, and ORF3). ORF3 encodes two capsid viral proteins (VP; VP1 and VP2). VP1 (75 kDa) and VP2 (61 kDa) share a C-terminal end. VP2 region locates in the crystal structure, from residues 39 to 536 [6,7]

Methods

Results

Conclusion