Digital recombinase polymerase amplification in hydrogel nanofluidic chip for ultrafast and precise quantification of pathogens in fresh food

The fast and precise point-of-care quantification of pathogens is an effective way to monitor and control diseases. In this work, we report a digital recombinase polymerase amplification (dRPA) in hydrogel nanofluidic chip for absolute quantification of nucleic acids as short as 5 min, without the need for complex chip operation. To perform the hydrogel RPA (gRPA), the RPA mixture with hydrogel monomers was introduced into a chip for cross-linking, followed by 39 °C amplification, resulting in a series of dots for digital counting. Since traditional Poisson distribution theory was no longer applicable here, a novel “Random Overlapping Theory” was developed for the first time for gRPA to calibrate the precise number of nucleic acids. With simple operation, the developed gRPA avoids pre-amplification during sample loading and partition. The detection performance of the nanofluidic system was analyzed by testing Listeria monocytogenes, confirming excellent quantitative ability with the limit of detection down to a single copy. This method is also demonstrated to possess excellent performance in real complex fresh food. Due to the facile operation, short detection time, low cost and low temperature required, as well as excellent quantification ability, we believe that the developed method will play an important role in point-of-care tests, especially in resource-limited areas.