Highly Sensitive\nOne-Pot Isothermal Assay Combining\nRolling Circle Amplification and CRISPR/Cas12a for Aflatoxin B<sub>1</sub> Detection

Occurrences of mycotoxins in cereals are widespread throughout\nthe world. However, the lack of efficient and ultrasensitive tests\nhas largely impeded the identification of these substances in actual\nsamples. Herein, a novel one-pot isothermal assay that integrates\nrolling-circle amplification (RCA) and CRISPR/Cas12a to detect aflatoxin\nB<sub>1</sub> (AFB<sub>1</sub>) is reported. Upon addition of AFB<sub>1</sub> to the magnetic bead functionalized with a duplex of the\nAFB<sub>1</sub> aptamer and its complementary DNA (cDNA), the specific\nrecognition of AFB<sub>1</sub> by the aptamer causes the release of\ncDNA to activate the RCA reaction. Subsequently, the RCA amplicon\ninitiates both trans-cleavage and cis-cleavage activities of the endonuclease\nCas12a. The synergistic coupling of RCA and CRISPR/Cas12a enables\nexponential amplification of cDNA, which further promotes CRISPR/Cas12a\nto nonspecifically cleave the single-stranded DNA reporters with enhanced\ndetection signals. Remarkably, the CRISPR/Cas12a-assisted one-pot\nisothermal assay can not only achieve ultrasensitive quantitative\ndetection through fluorescence detection, but also achieve visual\ndetection through a lateral flow strip, which improves accessibility\nto mycotoxin detection in resource-limited regions. The limit of detection\nwas 0.016 and 0.408 ng/mL, respectively. The proposed assay successfully\napplies in real samples with satisfactory recoveries from 90 to 114%.\nThis study presents a powerful and versatile method for reliable and\nultrasensitive detection of mycotoxins in various applications.