Development of a TaqMan RT-qPCR for the detection and genotyping of bovine viral diarrhea virus types 1, 2, and HoBi-like pestivirus.

内容摘要

HoBi-like pestivirus (HoBiPeV), as a newly discovered virus species in recent years, poses a serious threat to the global cattle industry due to its wide transmission and high pathogenicity. The genetic sequence of HoBiPeV exhibits significant differences when compared to the known strains of BVDV-1 (Pestivirus bovis) and BVDV-2 (Pestivirus tauri).Current detection methods are typically limited to identifying BVDV-1 and BVDV-2, and are unable to effectively differentiate HoBiPeV from the other two genotypes. To facilitate the rapid detection and differentiation of various genotypes, this study developed a pair of universal primers along with three TaqMan probes based on the 5' UTR sequences of the BVDV-1, BVDV-2, and HoBiPeV available in GenBank. A one-step multiplex TaqMan RT-qPCR detection method was developed. The results showed that this method could specifically detect the three genotypes of BVDV without cross-reactivity. It exhibited high sensitivity, with a minimum detection limit of 1 × 10¹copies/µL. The amplification efficiencies were 104.6%, 102.9%, and 117.7%, respectively, demonstrating a strong linear relationship (R2 > 0.99). This method also showed excellent intra-assay and inter-assay reproducibility, which facilitates accurate identification and detection of the three genotypes. In clinical sample testing, the present assay showed higher detection rates for all three genotypes than previously reported methods, particularly showing a notable sensitivity advantage in the detection of BVDV-1. This method not only significantly enhances the detection rate of HoBiPeV but also reveals the potential prevalence of this genotype in cattle populations, providing valuable insights for updating existing epidemiological databases and informing the selection of vaccine strains. The method is applicable in areas such as the detection of raw materials and intermediates during vaccine production, quality control of dairy products, and large-scale virus monitoring. It also exhibits broad application value in epidemiological investigations and the prevention and control of viral diseases in cattle farms.