Pronounced Fluorescence Polarization Enhancement Driven by RPA-CRISPR/Cas12a Induced Nucleoprotein Assembly for Salmonella Analysis in Animal-Derived Food Matrices

Salmonella is one of the most hazardous foodborne pathogens, posing a serious threat to public health and food safety worldwide. Conventional recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection assays predominantly rely on the trans-cleavage of fluorescent reporters; however, such signal-generation modes are inherently susceptible to photobleaching, signal drift, and fluctuation, thereby compromising quantitative accuracy and long-term signal stability in practical pathogen detections. To overcome these limitations, we developed a trans-cleavage-independent fluorescence polarization (FP) sensing platform for the rapid and quantitative detection of Salmonella . Unlike conventional reporter-cleavage-based readouts, the proposed system exploits target-induced nucleoprotein assembly to achieve direct, physical signal amplification. In this design, a FAM-labeled forward primer serves as an intrinsic molecular reporter, while exonuclease I (Exo I) selectively degrades unincorporated primers, effectively suppressing background interference. Upon recognition of Salmonella genomic DNA, RPA produces rigid double-stranded amplicons that restrict fluorophore rotational freedom, and subsequent crRNA-guided Cas12a binding further increases molecular size and hydrodynamic volume, resulting in a stepwise enhancement of FP signals. The assay exhibits excellent linearity over a concentration range of 3 × 10 1 –3 × 10 6 CFU mL –1, with an ultralow detection limit of 5 CFU mL –1 . In addition, it demonstrates outstanding photostability, reproducibility, and high specificity against nontarget bacteria. Importantly, reliable Salmonella detection was achieved in complex food matrices, including meat, eggs, and dairy products, with consistently high recoveries and strong tolerance to matrix interference, offering a promising alternative to conventional fluorescence-intensity-based CRISPR diagnostics in complex food systems.