CRISPR/Cas12a-mediated electrochemical biosensor for the sensitive detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus, a common gram-negative halophilic bacterium found in marine and estuarine environments, is a major cause of foodborne illnesses. This study presents the development of a highly sensitive and selective electrochemical DNA biosensor by combining the polymerase chain reaction (PCR) with the clustered regularly interspaced short palindromic repeats CRISPR-Cas12a (E-CRISPR) technology. The unique cis- and trans-cleavage activities of CRISPR-Cas12a were harnessed in the design of a hairpin DNA (hpDNA) probe, which was immobilized onto a gold electrode. This hpDNA probe enabled efficient cleavage and significantly improved detection sensitivity. Specifically, the hpDNA probe was cleaved from the electrode surface by Cas12a owing to its trans-cleavage activity, which resulted in a significant reduction in the current that was quantified to determine the detection result. The biosensor achieved a limit of detection (LOD) of 0.93 ng/mu L for the target DNA under optimal conditions. It also demonstrated a highly sensitive detection of V. parahaemolyticus within a linear range of 3.7 x 101 to 3.7 x 105 CFU/mL, exhibiting an LOD of 1.46 CFU/mL in a spiked mussel tissue matrix. The developed E-CRISPR biosensor, combined with PCR amplification, demonstrated high specificity, stability, and reproducibility. Compared to conventional PCR methods, this novel approach offers significant advantages for detecting V. parahaemolyticus containing tdh virulence gene. These advantages include enhanced specificity, faster analysis times, and clear, reliable results. Furthermore, the developed E-CRISPR biosensor has the potential to become a versatile and powerful tool for rapid and reliable diagnostics in public health and food safety monitoring.