Establishment and application of a visual Nfo-based recombinase polymerase amplification-lateral flow device assay for rapid detection of five bovine viruses (BVDV, AKAV, BNoV, BEV, and BCoV)

The cattle industry has incurred significant economic losses due to infections with various contagious diseases such as Bovine Viral Diarrhea (BVD), Akabane disease (AKA), Bovine Norovirus (BNoV), Bovine Enterovirus (BEV), and Bovine Coronavirus (BCoV). These pathogens often cause similar clinical manifestations, including diarrhea, vomiting, and reproductive disorders in pregnant cattle, and often occur as subclinical or mixed infections, complicating diagnosis. Therefore, the establishment of a rapid, sensitive, and field-deployable diagnostic assay is crucial for effective disease surveillance and control. The nfo RPA detection kit utilizes recombinase polymerase amplification combined with a lateral flow device (LFD) for visual interpretation of amplification products. The reaction system incorporates endonuclease IV (nfo), an nfo probe, and a biotin-labeled downstream primer. In this study, we developed a multiplex visual nfo RPA assay for the simultaneous detection of five bovine viruses (BVDV, AKAV, BNoV, BEV, and BCoV). Optimal primers for the nfo RPA assay were screened, and key reaction parameters (reaction time and temperature) were optimized. The established assay demonstrated high specificity, sensitivity, and repeatability. The optimal reaction time and temperature were determined to be 20 min and 39 °C, respectively. The limits of detection (LOD) were 10 1 copies/µL for BVDV, 10 2 copies/µL for AKAV, BNoV and BCoV, and 10 3 copies/µL for BEV. A total of 200 clinical samples collected from 10 farms in certain regions of Jilin Province, China, were tested. The results indicated that the nfo RPA-LFD assay was more intuitive and rapid. The visual nfo RPA assay created in this research is suitable for the efficient detection and analysis of bovine viruses, and provides fundamental technical support for the on-site diagnosis and control of specific bovine viral diseases.