Development of a Recombinase-Aided Amplification Assay for Rapid Detection of Human Norovirus GII.4

Background: Human noroviruses are one of the main causes of foodborne illnesses and represent a serious public health concern. Rapid and sensitive assays for human norovirus detection are undoubtedly necessary for clinical diagnosis, especially in regions without more sophisticated equipment. Method: The recombinase-aided amplification (RT-RAA) is a fast, robust and isothermal nucleic acid detection method based on enzyme reaction. This method can complete the sample detection at 39°C in 30 minutes. In this study, we successfully established a rapid reverse transcription recombinase-aided amplification (RT-RAA) assay for the detection of human norovirus GII.4 and applied this assay to clinical samples, as well as comparison with commercial RT-qPCR. Results: An analytical sensitivity detection of the RT-RAA at a 95% probability of 3.425 log10 genomic copies (LGC)/reaction. Moreover, no cross-reaction was observed with other norovirus genogroups and other common foodborne viruses. Stool samples were examined by RT-RAA and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Compared with RT-qPCR, kappa values for human norovirus detection with RT-RAA were 0.894 (p < 0.001), indicating that both assays were in agreement. Conclusion: This RT-RAA assay provides a rapid, specific, and sensitive assay for human norovirus detection and is suitable for clinical testing.