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.

CRISPR/Cas System: The Accelerator for the Development of Non-nucleic Acid Target Detection in Food Safety

“…A new analytical method has been reported for the ultrasensitive detection of citrinin according to CRISPR/ Cas12a (Figure 4C). 70 The probe was composed of AuNPs modified with antigen and thiol-terminated ssDNA. There was a competition between binding of antigen-modified AuNPs and citrinin to an anti-citrinin antibody which was coated with magnetic beads.…”

“…A new analytical method has been reported for the ultrasensitive detection of citrinin according to CRISPR/Cas12a (Figure C) . The probe was composed of AuNPs modified with antigen and thiol-terminated ssDNA.…”

Machine Learning-Mediated Ultrasensitive Detection of Citrinin and Associated Mycotoxins in Real Food Samples Discerned from a Photoluminescent Carbon Dot Barcode Array

“…Under propitious environmental conditions, CIT is found in cereals (rice, wheat, maize, gram, and barley), fruits (coffee, grapes, and apples), nuts (hazelnuts and Brazil nuts), and spices (cumin, turmeric, and black pepper) with damp storage conditions augmenting their presence . CIT causes nephrotoxicity, mitogenicity, genotoxicity, and hepatotoxicity upon ingestion that prompted several countries to set their limit of the presence in food for public health safety. , Consequently, CIT’s limit in rice is marked up to 100 μg/kg by the European Union, 50 μg/kg by China, and 200 μg/kg by Japan with intermediate values for many other countries . Along with CIT, the frequent synergistic presence of two other mycotoxins, namely, aflatoxin B1 (AFL) and ochratoxin A (OCT), makes the situation worse since the simultaneous detection of all three mycotoxins with a single detection strategy has never been achieved with conviction.…”