Double Isothermal Amplification and CRISPR-Cas12a for Sensitive Detection of Citrinin

Abstract

An analytical method is developed for ultrasensitive detection of citrinin using double isothermal amplification and CRISPR-Cas12a. Gold nanoparticles (AuNPs) modified with antigen and thiol-terminated, single-strand DNA (ssDNA) are used as a probe. The antigen-modified AuNPs compete with citrinin to bind to magnetic beads coated with an anticitrinin antibody. After a simple magnetic separation, the AuNPs are collected, and the ssDNA are released after they are washed with a dithiothreitol solution. The ssDNA is first amplified by an exponential amplification reaction and then used as a primer in a subsequent hybridization chain reaction to produce double-stranded DNA (dsDNA) that contains a protospacer adjacent motif to allow recognition by CRISPR-Cas12a. The dsDNA activates the Cas12a-gRNA to cleave a reporter ssDNA to generate a fluorescence signal. The developed analytical method has a low detection limit (0.127 ng mL–1) and a wide linear range (0.005–500 μg mL–1) for detection of citrinin. For detection of citrinin in oat and flour, recoveries of 97–104% and 105–111% are obtained, respectively. By combining double isothermal amplification with CRISPR-Cas12a, ultrahigh sensitivity and selectivity can be achieved for detection of toxins in food.