Enzyme-free catalytic hairpin assembly reaction-mediated micro-orifice resistance assay for the ultrasensitive and low-cost detection of Listeria monocytogenes

The rapid, reliable, ultra-sensitive, and cost-saving detection of Listeria monocytogenes (L. monocytogenes) has substantial implications for food safety. Thus, we developed a novel, enzyme-free, dual-signal amplification approach to detect L. monocytogenes based on the micro-orifice resistance technique combined with the aggregation of polystyrene (PS) microspheres constructed by the catalytic hairpin assembly reaction (CHA). Both the detection probes (probe2) and trigger DNA (tDNA) were first modified on PS microspheres (probe2-PS-tDNA). The tDNA was enriched by PS microspheres for the first signal amplification. After the hybridization reaction (the capture probes (probe1), target DNA, and probe2), unreacted probe2-PS-tDNA was removed, and the complex triggered the CHA reaction for the second signal amplification. Additionally, the micro-orifice resistance technique can sensitively identify PS microsphere aggregation caused by the CHA reaction to analyze the target DNA quantitatively. The CHA-mediated micro-orifice resistance assay was constructed by combining cost-saving PS microsphere probes, the highly specific DNA hybridization reaction, and the enzyme-free signal amplification strategy, substantially reducing the cost and improving the detection sensitivity (the limit of detection is 4 CFU/mL). This study provides a superior means to detect L. monocytogenes in complex food samples.