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 (TCID50) 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.IMPORTANCEBovine 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 TCID50 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.