Development of an on-site real-time dual detection method for norovirus and rotavirus using RPA-CRISPR/Cas12,13

Norovirus (NoV) and rotavirus (RoV) are the two most prevalent foodborne viruses responsible for infectious gastroenteritis, with high contamination rates in fruits and vegetables. Monitoring and accurately identifying these pathogens are crucial for preventing human foodborne illnesses. Therefore, developing a rapid, highly specific, and sensitive detection method for NoV and RoV is essential. An on-site real time dual detection method for Noroviruses of genogroup I (GI-NoV) and Rotavirus of group A (A-RoV) was established by integrating Recombinase Polymerase Amplification (RPA) with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (Cas) system. The DNA amplification products generated by multiplex RPA were simultaneously detected by Cas12a and Cas13a in a single tube. Utilizing the trans-cleavage preferences of Cas12a and Cas13a, the specific cleavage of DNA and RNA probes carrying different fluorescent groups within the reaction system was achieved. The results indicated that the established dual detection method had a detection limit of 10² copies/μL for GI-NoV and A-RoV. Specificity assay showed the established method only detected GI-NoV and A-RoV, with no cross-reactivity with other four foodborne viruses. Furthermore, the detection capability of this method was validated in strawberries and lettuce. The method enables the simultaneous detection of GI-NoV and A-RoV in fresh produce at 37 °C within 60 min. Detection results were interpreted by measuring fluorescence values or observing color changes under blue light at the end of the reaction. This technique required minimal instrumentation, provided excellent visual representation of results, and served as a technical reference for the rapid on-site detection of foodborne viruses in food matrices.