Establishment of duplex multi-enzyme isothermal rapid amplification detection method for bovine astrovirus and norovirus.

In recent years, the detection rates of bovine astrovirus (BoAstV) and bovine norovirus (BNoV) in Chinese farms have continued to rise, resulting in significant economic losses to the livestock industry. To meet the need for rapid on-site screening, this study established a dual nucleic acid detection system capable of simultaneously detecting BoAstV and BNoV using the multi-enzyme isothermal rapid amplification technology. Its specificity, sensitivity, and repeatability were further validated using clinical samples. The experimental results showed that the established MIRA method specifically amplified the target nucleic acids of BoAstV and BNoV, while exhibiting no cross-reaction with other common diarrhea-associated pathogens, such as bovine rotavirus (BRV), bovine viral diarrhea virus, and bovine coronavirus, indicating good specificity. The method demonstrated high sensitivity, with detection limits of 920 copies/μL for BoAstV and 6,100 copies/μL for BNoV. In the repeatability tests, all the coefficients of variation were below 10%, confirming the high stability of the method. In clinical sample testing, the method showed strong agreement with conventional dye-based quantitative PCR results (Kappa value = 0.887), while offering shorter detection time and higher detection rates. In summary, the detection method established in this study demonstrated high sensitivity, strong specificity, and a short turnaround time, making it suitable for the rapid detection of bovine astrovirus and bovine norovirus in clinical samples of cattle.