An electrochemical sensing method based on CRISPR/Cas12a system and hairpin DNA probe for rapid and sensitive detection of Salmonella Typhimurium

Abstract

Salmonella Typhimurium (S. Typhimurium) is regarded as a major cause of foodborne diseases, which has been identified as a server threat to public health. Herein, an electrochemical sensing method was developed to detect S. Typhimurium in combination with polymerase chain reaction (PCR) and CRISPR/Cas12a (E-CRISPR biosensor). The target DNA extracted from the bacteria was amplified by PCR first, followed by the second amplification through the activation of collateral cleavage activity of Cas12a against signaling hairpin DNA probes, causing the release of electrochemical labels and a dramatic current decrease. The design of hairpin DNA on the electrode also reduced the steric hindrance effect for better Cas12a collateral cleavage efficiency, leading to improved detection performance. Under the optimized conditions, the proposed E-CRISPR biosensor allowed the sensitive detection of S. Typhimurium in a linear range from 6.7 × 101 to 6.7 × 105 CFU/mL with a limit of detection of 55 CFU/mL in pure culture. In addition, the E-CRISPR biosensor enables the detection of S. Typhimurium in spiked poultry meat with excellent accuracy and sensitivity. This CRISPR-based biosensor has the potential to provide an alternative way for the detection of foodborne pathogens in the food supply chain.