Microfluidic integrated nucleic acid test (MINT) enabled by coupling FEN1-aided RPA with wax-purification and...

To guide timely antibiotic treatment, a tool for quick antimicrobial resistance gene detection is required. Here a Microfluidic Integrated Nucleic Acid Test platform (namely, MINT) was proposed for point-of-care molecular diagnostics. The platform integrates fast nucleic acid extraction based on hybrid wax-phase purification, and DNA amplification based on our newly developed FEN1-aided RPA (FARPA) into a microfluidic chip. The wax-phase stays solid at room temperature, while becomes liquid at >50 °C to allow the wash of inhibitors carried from extraction buffer, thus the chip keeps stable during storage and transportation. The movement of extracted DNA bound with magnetic beads were controlled by a magnet. The beaded DNA was quickly purified during the flow through the wax phase, and the amplification started once the beaded DNA reached to the well for FARPA. The signal from target-positive sample could be readily sensed by a conventional smartphone. The turnaround time is about 40 min from the sample to the result. The platform was validated by detecting the blaKPC-2 resistance gene of Klebsiella pneumoniae (K. pneumoniae) from urine samples with the reliable detection concentration of 500 CFU/mL, achieving 100% agreement for the 29 clinical urine samples (19 positive, 10 negative) with conventional qPCR. It is easy to adapt the MINT to any other pathogen only by designing the corresponding primers and probes for FARPA. It is promising to apply MINT for bed-site antimicrobial resistance screening just after operation.