Journal
ANALYTICAL CHEMISTRY
Volume 93, Issue 42, Pages 14300-14306Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c03468
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Funding
- National Natural Science Foundation of China [31901785, 32172307, 32172285, 31701683]
- Basic Public Welfare Research Program of Zhejiang Province [GN21C200026]
- Natural Science Foundation of Fujian Province, China [2019J01239]
- Key Research and Development Program of Zhejiang Province, China [2021C02062]
- Open Fund of State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products [2010DS700124-KF2009]
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A novel label-free, colorimetric method for visual detection of V. parahaemolyticus was proposed, utilizing CRISPR/Cas12a to trim G-quadruplex DNAzyme and prevent color development of ABTS(2-). The method showed sensitivity comparable to real-time LAMP, detecting 6.1 x 10^2 CFU/mL V. parahaemolyticus in shrimp samples, making it a promising universal biosensing strategy for field pathogenic bacterial testing.
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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