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
A CbAgo-CRISPR/Cas12a-powered cascade amplification sensing strategy was proposed. • It combined CbAgo's precise cleavage with the Cas12a's robust trans-cleavage activity. • An amplified fluorescent biosensor was developed for detecting Salmonella Typhimurium . • The proposed biosensor demonstrated comparable analytical performance to qPCR assay. Sensitive and accurate detection of foodborne pathogens constitutes a critical imperative for safeguarding food safety. Herein, we proposed a Clostridium butyricum argonaute (Cb A go)-CRISPR/ C as12a-powered synergistic amplification strategy and developed a DNA amplification-free f luorescent ( ACF ) biosensor for ultrasensitive detection of Salmonella Typhimurium . In this biosensor, magnetic beads-phage conjugates enabled the selective capture of viable Salmonella Typhimurium , followed by rapid DNA extraction within 5 min using commercial lysis buffers. A synergistic signal amplification strategy was designed for efficient biosensing by utilizing the primary cleavage product (tDNA) of the CbAgo system as a molecular bridge to integrate CbAgo and Cas12a systems. Specifically, CbAgo performed primary cleavage of target DNA to generate tDNA fragments. These tDNA subsequently activated the CRISPR/Cas12a system, initiating its trans-cleavage activity to degrade fluorescent reporter probes for quantitative analysis. This tDNA-mediated integration synergizes CbAgo's precision cleavage capability with Cas12a's efficient trans-cleavage activity, enabling exponential amplification. Under optimized conditions, the ACF biosensor exhibited a linear detection range of 10 2 –10 7 CFU/mL and a detection limit of 24.8 CFU/mL for Salmonella Typhimurium without DNA amplification steps. Moreover, the ACF biosensor demonstrated comparable analytical performance to the qPCR assay and achieved satisfactory results in the detection of Salmonella Typhimurium in 20 food samples, highlighting its potential as an ultrasensitive tool for foodborne pathogen monitoring. Download: Download high-res image (107KB) Download: Download full-size image
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