Exponential isothermal amplification-assisted Clostridium butyricum argonaute-based biosensor for homogeneous, multiplex, and sensitive detection of foodborne pathogens

Due to the significant health risks posed by pathogenic bacteria, there is an urgent need to develop multiplex, sensitive detection methods for them. Here, we developed a highly sensitive fluorescent sensor for the detection of pathogens by leveraging the powerful programmability of Clostridium butyricum Argonaute ( Cb Ago)-assisted exponential isothermal amplification reaction (EXPAR). This system combined Cb Ago, which locally cleaved extracted genomic DNA to produce single-stranded DNA (ssDNA), with EXPAR, which induced the amplification of large DNAzyme fragments by the ssDNA. Each DNAzyme fragment cleaved a specific reporter to generate a fluorescent signal for quantification of pathogenic bacteria. The EXPAR templates were carefully designed to ensure sufficient specificity for multiplex detection. The assay demonstrated superior analytical performance, with a limit of detection of as low as 4 CFU mL −1 and a linear range of 10–10 7 CFU mL −1 . This method represents the integration of Cb Ago-mediated genome editing and EXPAR high-efficiency amplification, yielding a customizable nucleic acid detection tool with broad applications in pathogen detection