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

Man Zhang, Xiaoting Xue, Haiyue Gong, Baolin Liu, Lei Ye

Research output: Contribution to journalArticlepeer-review


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.
Original languageEnglish
Pages (from-to)1997-2005
Number of pages9
JournalACS Agricultural Science and Technology
Issue number10
Publication statusPublished - 2021 Oct 25

Subject classification (UKÄ)

  • Analytical Chemistry
  • Biochemistry and Molecular Biology

Free keywords

  • Ultrasensitive detection
  • Biobarcode
  • CRISPR-Cas12a
  • Exponential amplification reaction
  • Hybridization chain reaction
  • Citrinin


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