CRISPR/Cas12a-mediated cyclic signal amplification and electrochemical reporting strategy for rapid and accurate sensing of Vibrio parahaemolyticus in aquatic foods

Rapid and accurate detection of target foodborne pathogenic bacteria is extremely important for preventing and controlling foodborne diseases. Vibrio parahaemolyticus (V. parahaemolyticus, Vp) is considered as a major cause of foodborne diseases, posing severe threat to food safety and public health. The efficiency and sensitivity of traditional protocols for Vp identification is time consuming and of poor precision. In this research, a simple electrochemical sensing method was developed for accurate detection of Vp in aquatic products. Target genes of Vp were rapid amplified with the designed recombinase polymerase amplification, which further activated the designed CRISPR/Cas12a system. The electrochemical active ssDNA probe on the sensing interface would be hydrolyzed by the activated trans-cleavage activity of Cas12a, inducing the release of active electrochemical tags from the sensing interface and the decreased sensing signals. Under the optimized conditions, this proposed RPA-mediated electrochemical-CRISPR (E-CRISPR) biosensor enabled sensitive detection of target Vp over a linear range from 10¹ to 10⁶ CFU/mL, with limit of detection of 32 CFU/mL. Additionally, this E-CRISPR biosensor realized the successful determination of Vp in spiked fish samples with satisfied sensing performance. The isothermal amplification and the rapid electrochemical response of the E-CRISPR biosensor made it suitable for on-site screening. And this E-CRISPR biosensor could be well integrated with other isothermal protocols and extended to other target pathogens, showing great potential for practical applications in molecular diagnostics and other gene detection related fields.