Статья

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

Xueyun ChenCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaLiu WangState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Key Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs; Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, ChinaFang HeState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Key Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs; Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, ChinaGanghui ChenCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaLinlin BaiCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaKaiyu HeState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Key Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs; Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, ChinaFang ZhangCollege of Biological Science and Engineering, Fuzhou University, Fuzhou 350108, ChinaXiahong XuState Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products; Key Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture and Rural Affairs; Institute of Agro-product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

2021en

ABI

Аннотация

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) (ABTS2–), which displays colorless for positive samples while green for target-free samples. We term such Cas12a–crRNA preventing ABTS2– 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.

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Идентификаторы

DOI: 10.1021/acs.analchem.1c03468

Цитирования и источники

Цитирований: 2Использованных источников: 0

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