CRISPR-Based Colorimetric Nucleic Acid Tests for Visual Readout of DNA Barcode for Food Authenticity

Abstract

Food authenticity is a critical issue associated with the economy, religion, and food safety. Herein, we report a label-free and colorimetric nucleic acid assay for detecting DNA barcodes, enabling the determination of food authenticity with the naked eye. This method, termed the CRISPR-based colorimetric DNA barcoding (Cricba) assay, utilizes CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR associated protein) to specifically recognize the polymerase chain reaction (PCR) products for further trans-cleavaging the peroxidase-mimicking G-quadruplex DNAzyme. Based on this principle, the presence of the cytochrome oxidase subunit I gene could be directly observed with the naked eye via the color change of 3,3′,5,5′-tetramethylbenzidine sulfate (TMB). The whole detection process, including PCR amplification and TMB colorimetric analysis, can be completed within 90 min. The proposed assay can detect pufferfish concentrations diluted to 0.1% (w/w) in a raw pufferfish mixture, making it one of the most sensitive methods for food authenticity. The robustness of the assay was verified by testing four common species of pufferfish, including Lagocephalus inermis, Lagocephalus spadiceus, Takifugu bimaculatus, and Takifugu alboplumbeus. The assay is advantageous in easy signal readout, high sensitivity, and general applicability and thus could be a competitive candidate for food authenticity.

Recent progress on the CRISPR/Cas system in optical biosensors

“…As mentioned previously, the CRISPR/Cas-based colorimetric biosensors have significant potential for rapid on-site detection in food testing, 75,88 clinical diagnoses, 80,89 and environmental monitoring. 76 Nonetheless, limited color contrast remains challenging to achieve accurate quantification.…”

“…74 Yin et al proposed a peroxidase-mimicking G-quadruplex DNAzyme-based colorimetric DNA barcoding (Cricba) platform to detect food authenticity. 75 As shown in Fig. 3A, in the presence of target genes, the G-quadruplex underwent cleavage by the activation of Cas12a, leading to the inhibition of its peroxidase-like activity to consequently impede the catalysis of TMB.…”