Rapid two‐stage amplification in a single tube for simultaneous detection of norovirus GII and group a rotavirus

The most prevalent viruses currently causing diarrhea are norovirus and rotavirus, and rapid and sensitive detection methods are essential for the early diagnosis of disease. The purpose of this study was to establish a sensitive single‐tube two‐stage nucleic acid amplification method—reverse transcription recombinase‐assisted PCR (RT‐RAP)—for simultaneous detection of norovirus GII and group A Rotavirus, with the first stage consisting of isothermal reverse transcription recombinase‐aided amplification (RT‐RAA) and the second stage consisting of qPCR (quantitative PCR). RT‐RAP is more sensitive than either RT‐RAA or qRT‐PCR (quantitative RT‐PCR) alone. And the addition of a barrier that can be disassembled after heating enabled the detection of samples within 1 h in a single closed tube. Sensitivity was 10 copies/reaction of norovirus (Novs) GII and group A rotavirus (RVA). In parallel, two hundred fecal specimens were used to evaluate the method and compare it with a commercial fluorescent quantitative RT‐PCR. The data showed kappa values of 0.957 and 0.98 (p < 0.05) for detecting Novs GII and RVA by the two methods, indicating the potential of the newly established assay to be applied to clinical and laboratory testing.