Rapid, sensitive, and visible RPA-LFD assay for BoHV-1 and BoHV-5

ABSTRACT Bovine herpesvirus (BoHV) infection poses a significant threat to the healthy development of the cattle industry. BoHV-1 primarily causes infectious bovine rhinotracheitis, while BoHV-5 is associated with bovine necrotic meningoencephalitis. These two pathogens not only exhibit a high correlation in antigenicity and genetic background but, more importantly, can establish latent infections within the bovine ganglion. Therefore, establishing a rapid and convenient pathogen detection method for BoHV-1 and BoHV-5 is crucial for early diagnosis, epidemic monitoring, and minimizing economic losses. In this study, utilizing the conserved sequences of BoHV-1 and BoHV-5 as targets, we developed a rapid and visually accessible recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay suitable for on-site detection. This method enables visual detection within 30 min for clinical samples containing as little as 1 tissue culture infectious dose 50 (TCID 50 ) and performs comparably to qPCR, with slightly lower sensitivity. The findings of this research offer new insights for early clinical detection and the control of BoHV-1 and BoHV-5 infections. IMPORTANCE Bovine herpesvirus (BoHV), a significant respiratory pathogen in cattle, is widely prevalent worldwide, posing a substantial economic threat to the cattle industry. In this study, we have developed a rapid on-site detection method for BoHV, termed RPA-LFD, which can carry out pathogen detection with a sensitivity of no less than 1 TCID 50 within 30 min. When combined with screening, isolation, and culling measures, this approach can effectively eliminate the sources of BoHV infection, providing a scientifically feasible solution for BoHV prevention and control.