Development of a novel RAA-based microfluidic chip for absolute quantitative detection of human norovirus

As the rapid spread of norovirus is a major public health problem, rapid diagnostic assays are essential in assisting the implementation of appropriate control measures. Here, we presented a facile sample partitioning norovirus digital isothermal detection (NoV-DID) chip that enables the application of a fast and low-cost digital nucleic acid amplification test (dNAAT) to norovirus detection. Our microfluidic chip had 3 layers, a PDMS cover layer, a functional layer, and a supporting layer, and consisted of a total of 5120 microchambers, each of which was able to contain a volume of 1.2 nL. This chamber digital reverse transcription recombinase-aid amplification (cdRT-RAA) assay achieved equal volume partition of samples within 10 min, with the signal amplification being completed in approximately 20 min at 39 °C, without the need for a thermal cycle device. The cdRT-RAA assay showed a good degree of linearity in the range of 1.02 × 100 to 2.04 × 103 copies (R2 = 0.99) with the limit of detection being 1.02 × 100 copies/μL, demonstrating that its sensitivity was comparable to that of RT-ddPCR. The digital nucleic acid detection method based on the microfluidic chip exhibited advantages of high sensitivity and high robustness, and might provide an alternative method for the early detection of norovirus and the prevention and control of epidemics.