Rapid on-site and simultaneous detection of four foodborne Vibrio species in seafood using a microfluidic RPA-CRISPR/Cas12a platform.

Foodborne Vibrio species commonly contaminate seafood and often co-occur, complicating rapid on-site detection. We developed a centrifugal microfluidic chip integrating one-pot RPA-CRISPR/Cas12a for simultaneous detection of four key species: V. parahaemolyticus, V. vulnificus, V. cholerae, and V. alginolyticus. In this system, target-specific RPA amplification generates amplicons that activate Cas12a-mediated trans-cleavage of fluorescent ssDNA reporters, producing target-dependent fluorescence signals for pathogen identification. The chip has eight units, each with four reaction chambers with lyophilized target-specific reagents, enabling a simple "DNA-in, result-out" workflow. Operating at 39 °C, the assay is completed within 60 min in a closed system to minimize contamination. It shows high specificity with no cross-reactivity. Limits of detection are 100 copies/μL for V. parahaemolyticus and V. vulnificus, and 101 copies/μL for V. cholerae and V. alginolyticus. Performance in spiked and real shrimp samples matched qPCR, with 100% sensitivity and specificity. The portable platform costs about $3.29 per test. This platform offers a practical approach for on-site multiplex screening of pathogenic Vibrio in seafood.