Development of a Label-Free Colorimetric and Fluorescent Diagnostic Platform for Foodborne <i>Salmonella</i> Based on RPA-CRISPR/Cas12 Assay in a Single Tube

Foodborne pathogen <i>Salmonella</i> poses a significant threat to public health, and therefore, it is important to establish accurate, convenient, and rapid detection methods. Herein, a label-free colorimetric and fluorescent diagnostic platform for foodborne <i>Salmonella</i> was developed, integrating recombinase polymerase amplification (RPA), CRISPR/Cas12, and water-soluble cationic conjugated polythiophene (PMNT) in a single-tube system. Upon recognition of <i>Salmonella</i>-specific gene <i>invA</i> targets, RPA products can stimulate the cis- and trans-cleavage activity of Cas12a in the presence of crRNA. This enzymatic activity degrades single-stranded DNA (ssDNA), leading to the release of PMNT from PMNT-ssDNA complexes, which, in turn, produces a detectable fluorescence increase along with a visible color transition from pink to yellow. The one-tube strategy enables sensitive detection of 1.9 × 10¹ copies/μL <i>invA</i> target and could detect as low as 10³ CFU/mL of <i>Salmonella</i> in artificially spiked milk and fish samples without enrichment, while the detection limit improved to 10⁰ CFU/mL after 8 h enrichment. Importantly, the assay demonstrated high specificity with no cross-reactivity with other bacteria with and without complex food matrices. This one-tube, dual-signal assay provides a rapid, reliable, and equipment-minimal solution for on-site detection of <i>Salmonella</i>, with a reduced risk of aerosol contamination.