Label-Free Colorimetric Method for Detection of Vibrio parahaemolyticus by Trimming the G-Quadruplex DNAzyme with CRISPR/Cas12a

Vibrio parahaemolyticus (V. parahaemolyticus), which may cause gastrointestinal disorders in humans, is a pathogen commonly found in seafood. There are many methods for detecting V. parahaemolyticus, yet they have some shortcomings, such as high cost, labor-intensiveness, and complicated operation, which are impractical for resource-limited settings. Herein, we present a sequence-specific, label-free, and colorimetric method for visual detection of V. parahaemolyticus. This method utilizes CRISPR/Cas12a to specifically recognize the loop-mediated isothermal amplification (LAMP) products for further trans-cleaving the G-quadruplex DNAzyme and depriving its peroxidase-mimicking activity. In this way, the results can be directly observed with the naked eyes via the color development of 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS^2-), which displays colorless for positive samples while green for target-free samples. We term such Cas12a-crRNA preventing ABTS^2- from developing color by trimming the G-quadruplex DNAzyme as Cascade. The proposed method can detect 9.8 CFU (per reaction) of pure cultured V. parahaemolyticus, and the sensitivity is comparable to real-time LAMP. It has been applied for practical use and showed the capability to detect 6.1 × 10² CFU/mL V. parahaemolyticus in shrimp samples. Based on this, the newly established Cascade method can be employed as a universal biosensing strategy for pathogenic bacterial testing in the field.