Ultrasensitive, portable and multiplexed molecular detection of pathogenic bacteria in a cross-validating manner via Argonaute-triggered and reverse-phase enhanced fluorescent lateral flow bioassay

Foodborne illnesses and contamination are becoming increasingly severe global public health issues. In recent years, the resistance of pathogenic bacteria has increased significantly, which makes it particularly urgent to develop advanced detection technologies beyond traditional methods. PfAgo (Pyrocus furiosus Argonaute), a novel programmable nuclease with specific sequence targeting and cleavage capabilities, offers significant advantages over traditional CRISPR diagnostics (CRISPR-Dx) in molecular detection, particularly in flexible design and multiplexed detection. In this work, PfAgo was innovatively integrated with the nucleic acid reverse-phase enhanced fluorescent lateral flow test strip (rLFTS) and smartphone for the first time, enabling ultrasensitive, portable and multiplexed molecular detection of pathogenic bacteria in a cross-validating manner. PfAgo precisely recognized LAMP amplicons, triggering linker DNA cleavage that generated dual colorimetric/fluorescent signals on rLFTS. A 3D-printed visualizer equipped with a homemade App-enabled smartphone achieved single-cell sensitivity (1 CFU/mL) within 45 min, two orders of magnitude lower than traditional qPCR (∼100 CFU/mL). In summary, a conceptually novel cross-validating manner integrating PfAgo with dual-mode rLFTS was constructed. It eliminated the reliance on large instruments, representing a significant step forward in developing an ultrasensitive, portable, accurate and multiplexed molecular detection technology for foodborne pathogens.