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

文献类型： 外文期刊

第一作者： Chen, Xueyun

作者： Chen, Xueyun;Chen, Ganghui;Bai, Linlin;Zhang, Fang;Chen, Xueyun;Wang, Liu;He, Fang;Bai, Linlin;He, Kaiyu;Xu, Xiahong;Chen, Xueyun;Wang, Liu;He, Fang;Bai, Linlin;He, Kaiyu;Xu, Xiahong;Chen, Xueyun;Wang, Liu;He, Fang;Bai, Linlin;He, Kaiyu;Xu, Xiahong

作者机构：

期刊名称：ANALYTICAL CHEMISTRY （ 影响因子：6.986； 五年影响因子：6.755 ）

ISSN： 0003-2700

年卷期： 2021 年 93 卷 42 期

页码：

收录情况： SCI

摘要： 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 Gquadruplex 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 x 10(2) 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.

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