Development and application of a dual RT-qPCR assay for the differential detection of bovine rhinitis virus genotypes A and B.

Bovine rhinitis virus (BRV) is an important pathogen causing bovine respiratory disease complex (BRDC). To facilitate the rapid and simultaneous detection of bovine rhinitis A virus (BRAV) and bovine rhinitis B virus (BRBV), specific primers and TaqMan probes targeting the viral 3D gene were designed and synthesized. Following the optimization of primer and probe concentrations and annealing temperatures, as well as comprehensive evaluations of specificity, sensitivity and reproducibility, a dual real-time RT-PCR (RT-qPCR) assay was successfully developed for the reliable differential detection of BRAV and BRBV. The results demonstrated that the developed assay effectively differentiates between BRAV and BRBV. Furthermore, the assay exhibited no cross-reactivity with other common bovine respiratory pathogens, including IDV, BVDV, BCoV, BRSV, BPIV3, IBRV, BAdV-3, M. bovis, M. haemolytica, P. multocida, C. perfringens, and K. pneumoniae, thereby indicating its high specificity. The in vitro transcribed viral 3D gene RNA was used as template, the limit of detection (LOD) for both BRAV and BRBV was 1.0 × 102 copies/μL. Standard curves generated by plotting the logarithmic RNA concentrations against the corresponding cycle threshold (Ct) values yielded R2 values of 0.994 and 0.999, respectively. Finally, both intra- and inter-assay coefficients of variation were less than 3%, demonstrating excellent reproducibility. A total of 790 bovine nasal swab samples were analyzed using the dual RT-qPCR assay, and the results revealed the viral nucleic acid positive rates of 3.79% (30/790) for BRAV and 9.36% (74/790) for BRBV, with a co-infection rate of 1.26% (10/790). The dual RT-qPCR assay developed for the differential detection BRAV and BRBV demonstrated high specificity, sensitivity, and reproducibility, thereby providing a robust technical support for the surveillance, epidemiological investigation, and targeted prevention and control of BRV infection in the cattle industry.