CRISPR/Cas12a-Based\nIndirect Competitive Enzyme-Linked\nImmunosorbent Assay for Sensitive Detection of Ochratoxin A

The high toxicity and widespread contamination of ochratoxin\nA\n(OTA) make it urgent to develop a sensitive method to detect trace\nOTA in complex food matrices. Herein, an indirect competitive enzyme-linked\nimmunosorbent assay (icELISA)-based on the CRISPR/Cas12a system is\ndescribed. DNA amplicons with multiple activation sequences of the\nCRISPR/Cas12a system were pre-prepared to improve detection sensitivity.\nIn the absence of OTA, streptavidin-mediated biotinylated DNA amplicons\nwere captured by the biotinylated secondary antibody on the microplate.\nThe captured DNA amplicons activated the CRISPR/Cas12a system, which\nthereby effectively cleaved the reporter DNA, producing strong fluorescence.\nThe presence of OTA led to a decrease in DNA amplicons on the microplate,\nresulting in a decrease in activated Cas12a and ultimately a drop\nin fluorescence intensity. OTA in food matrices at nanogram per milliliter\nlevels can be detected. Therefore, the new method has great potential\nin monitoring OTA.