Autocatalytic CRISPR/Cas12a-Enabled Fluorescence Polarization for Highly Sensitive Detection of Escherichia coli across Diverse Food Matrices

Abstract Rapid and sensitive detection of bacterial contamination in complex food matrices is essential for food safety monitoring. Here, we report an autocatalytic CRISPR/Cas12a-enabled fluorescence polarization (FP) assay for Escherichia coli (E. coli) detection. Following recombinase polymerase amplification (RPA), target amplicons activate Cas12a to cleave a switchable dual-loop reporter (SDL-Reporter). Cleavage releases small fluorescent fragments with enhanced rotational diffusion, decreasing FP, while simultaneously liberating functional crRNA that recruits additional Cas12a for recursive Cas12a activation. This dual-functional design couples feedback amplification with rotational diffusion-based FP transduction. The assay achieved a detection limit of 5 CFU/mL, a linear range of 1.8 × 101 to 1.8 × 107 CFU/mL, and high specificity toward E. coli. Evaluation in 11 pretreated food matrices covering fruits, vegetables, meats, and dairy products demonstrated satisfactory analytical accuracy and reproducibility across different food types. These findings support the potential of the proposed strategy for sensitive foodborne bacterial analysis.