Ultrasensitive detection of pathogenic bacteria by CRISPR/Cas12a coupling with a primer exchange reaction

The detection of pathogenic bacteria is extremely important to maintain food safety. In the present study, we constructed an electrochemical biosensor for point-of-care testing (POCT) of pathogenic bacteria through the cascade signal amplification of CRISPR/Cas12a and primer exchange reaction (PER) without nucleic acid extraction. Functional DNA aptamers locked the hairpin of PER, thereby preventing primer extension in the absence of target pathogenic bacteria. The presence of target pathogenic bacteria triggered the unlocking of the hairpin of PER and extended the primer into a long single-stranded DNA (ssDNA), which then activated the cleavage of ssDNA modified on an Au electrode by Cas12a, resulting in the decrease of the electrochemical signal detectable by an electrochemical workstation. The sensor could quantify Escherichia coli O157:H7 concentration from 10 to 106 CFU mL−1, with a detection limit of 19 CFU mL−1. Target bacteria in milk samples can be successfully detected by this method. On the basis of these characteristics, the proposed electrochemical biosensor based on CRISPR/Cas12a and PER could serve as a prospective tool for new point-of-care (POC) applications in food safety.