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

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.