Highly Sensitive One-Pot Isothermal Assay Combining Rolling Circle Amplification and CRISPR/Cas12a for Aflatoxin B₁ Detection

Occurrences of mycotoxins in cereals are widespread throughout the world. However, the lack of efficient and ultrasensitive tests has largely impeded the identification of these substances in actual samples. Herein, a novel one-pot isothermal assay that integrates rolling-circle amplification (RCA) and CRISPR/Cas12a to detect aflatoxin B₁ (AFB₁) is reported. Upon addition of AFB₁ to the magnetic bead functionalized with a duplex of the AFB₁ aptamer and its complementary DNA (cDNA), the specific recognition of AFB₁ by the aptamer causes the release of cDNA to activate the RCA reaction. Subsequently, the RCA amplicon initiates both trans-cleavage and cis-cleavage activities of the endonuclease Cas12a. The synergistic coupling of RCA and CRISPR/Cas12a enables exponential amplification of cDNA, which further promotes CRISPR/Cas12a to nonspecifically cleave the single-stranded DNA reporters with enhanced detection signals. Remarkably, the CRISPR/Cas12a-assisted one-pot isothermal assay can not only achieve ultrasensitive quantitative detection through fluorescence detection, but also achieve visual detection through a lateral flow strip, which improves accessibility to mycotoxin detection in resource-limited regions. The limit of detection was 0.016 and 0.408 ng/mL, respectively. The proposed assay successfully applies in real samples with satisfactory recoveries from 90 to 114%. This study presents a powerful and versatile method for reliable and ultrasensitive detection of mycotoxins in various applications.