Establishment and Application of Real-time Fluorescence Quantitative RT-PCR for Detection of BVDV Genotype 1

PurposeTo establish a real-time fluorescence quantitative RT-PCR for detection of bovine viral diarrhea virus of genotype 1 (BVDV-1).

MethodsThe specific primers and probes were designed based on the analysis of the genome sequence of the dominant BVDV-1 strains in past five years. Reaction system and condition of the real-time fluorescence quantitative PCR detection method were screened and optimized. The specificity, sensitivity, reproducibility, and clinical sample testing were conducted.

ResultsThe optimal probe concentration was 0.25 μmol/L and the primer concentration was 0.60 μmol/L. The minimum detection limit for positive template plasmid control was 1.7 copies/μL. The specificity assay showed the established method only specifically amplified BVDV-1, while no amplification of blue tongue disease virus, foot-and-mouth disease virus, vesicle stomatitis virus, peste des petits ruminants virus and swine fever virus. Good repeatability was obtained with a coefficient of variation less than 1%. Detection of 172 clinical blood samples using the established method, it revealed 33 BVDV-1 positive samples with a positive rate of 19.19%, which was consistent with the result obtained by real-time fluorescence quantitative RT-PCR in the national standard of the diagnostic techniques for bovine viral diarrhea/mucosal disease in China (GB/T 18637—2018).

ConclusionThe established method of real-time fluorescence quantitative PCR for detection of BVDV-1 is specific, sensitive and with a good repeatability. It can be used for detection and monitoring of BVDV-1 type, and provides a powerful technical means for detection and monitoring of BVDV-1