CRISPR-mediated recombinase-assisted amplification for hazard assessment of Vibrio parahaemolyticus on microfluidic system

Abstract

Highly sensitive and accurate detection of virulence and drug resistance genes in Vibrio parahaemolyticus ( V. parahaemolyticus ) is essential for disease prevention and the control of foodborne transmission. In this research, a novel RAA-CRISPR/Cas12a-based microfluidic hazard assessment platform (RCMHA) was developed for the identification of virulence genes tdh , trh and toxR , as well as drug resistance genes tetA , floR and sul1 of V. parahaemolyticus in seafood samples, thereby enabling hazard assessment based on the detection of hazard-associated genetic determinants. By integrating multi-channel spatial coding within an enclosed reaction, the centrifugal microfluidic chip mitigates the limited sensitivity caused by the interference between RAA amplification and Cas12a trans-cleavage in this system. Critical parameters of the RCMHA system were systematically optimized to maximize fluorescence signal output and detection efficiency, including the Cas12a/crRNA ratio, probe concentration, and RAA reaction. RCMHA system enables high-throughput simultaneous detection of all six target genes within 40 min, with the detection limits of 10 1 fg/µL. In addition, the system was successfully applied to detect artificially contaminated shrimp samples with high specificity. Therefore, the established RCMHA system offers a practical and efficient molecular detection method, with the potential for extension to other gene detection applications.