CRISPR/Cas12a-based simplified lateral flow assay for sensitive detection of OTA and AFB1

Mycotoxin contamination in agricultural commodities poses serious health risks, necessitating sensitive and user-friendly detection methods for food safety monitoring. Here, we present a multiplex CRISPR/Cas12a-assisted lateral flow assay (MC-LFA) for the parallel detection of Ochratoxin A (OTA) and Aflatoxin B1 (AFB1). The platform employs a duplex Switch design in which target binding releases an activator sequence (Probe 2), triggering Cas12a trans-cleavage of a half-blocking reporter (HBR). Cleavage generates dsDNA products labeled with Dig/FAM (for OTA) or TAMRA/FAM (for AFB1), which are captured at distinct test lines via specific antibody recognition. An integrated streptavidin (SA) capture line binds intact HBR through biotin-streptavidin interactions, effectively replacing conventional magnetic separation steps. The total assay time is approximately 65 min, including a 45-min target identification and a 15-min Cas12a reaction followed by a 5-min lateral flow readout. This approach achieves visual detection limits of 50 pg/mL for OTA and 250 pg/mL for AFB1. The assay exhibits excellent linear correlation over broad concentration ranges and demonstrates high accuracy in double-blind testing (97.22% for OTA and 94.44% for AFB1). Notably, the platform requires only aptamer replacement for different targets while maintaining the same Cas12a and crRNA sequences, substantially reducing experimental costs. Furthermore, by co-localizing detection probes in a single tube, an "OR" logic gate strategy enables parallel screening for both mycotoxins with a single test line. This cost-effective, sensitive, and operationally simple platform possesses strong potential for on-site food safety monitoring in resource-limited settings.