Multiplexed food-borne pathogen detection using an argonaute-mediated digital sensor based on a magnetic-bead-assisted imaging transcoding system

Accurate, sensitive and multiplexed detection of food-borne pathogens is crucial for assessing food safety risks. Here we present a digital DNA-amplification-free nucleic acid detection assay to achieve multiplexed and ultrasensitive detection of three food-borne pathogens. We used mesophilic Clostridium butyricum argonaute and magnetic beads in a digital carrier system (d-MAGIC). Clostridium butyricum argonaute, with its two-guide accurate cleavage activity, precisely targets and cleaves fluorescence-quencher reporters corresponding to different bacteria through a two-step process. The system uses fluorescence-encoded magnetic beads as programmable multi-probes, allowing the simultaneous detection of multiple pathogens and easy data interpretation via artificial intelligence. The method showed a wide detection range (10¹ to 10⁷ CFU ml^-1) and a low limit of detection of 6 CFU ml^-1 for food-borne pathogens without DNA amplification. Digital nucleic acid testing using d-MAGIC can become a next-generation strategy for accurate and convenient pathogen detection.