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A CRISPR/Cas12a-based label-free fluorescent method for visual signal output

文献类型： 外文期刊

作者： Wang, Liu 1 ; He, Fang 1 ; Chen, Xueyun 1 ; He, Kaiyu 1 ; Bai, Linlin 1 ; Wang, Qiang 1 ; Zhang, Fang 2 ; Xu, Xiahong 1 ;

作者机构： 1.Zhejiang Acad Agr Sci, Inst Agroprod Safety & Nutr, State Key Lab Managing Biot & Chem Threats Qual &, Hangzhou 310021, Peoples R China

2.Fuzhou Univ, Coll Biol Sci & Engn, Fuzhou 350108, Peoples R China

3.Minist Agr & Rural Affairs, Key Lab Informat Traceabil Agr Prod, Hangzhou 310021, Peoples R China

4.Ningbo Univ, Coll Food & Pharmaceut Sci, Ningbo 315800, Peoples R China

关键词： CRISPR/Cas12a; Visual detection; G-quadruplex; Thioflavin-T; Label-free; Vibrio parahaemolyticus

期刊名称：SENSORS AND ACTUATORS B-CHEMICAL （ 影响因子：9.221； 五年影响因子：7.676 ）

ISSN：

年卷期： 2022 年 370 卷

页码：

收录情况： SCI

摘要： CRISPR/Cas12a, as a powerful and programmable biosensing tool, has brought great convenience in visual detection of the target in a sequence-specific mode. Though fluorescent output is the predominant way of present visual methods, this strategy highly depends on cleaving fluorophore-/quencher-labeled oligonucleotides. Few research focuses on realizing fluorescent visual detection without DNA-labeling. Herein, we have proposed a CRISPR/Cas12a-based label-free fluorescent visual detection strategy. The basic principle of this strategy is that G-quadruplex can enhance the fluorescent emission of fluorescent ligands while dsDNA target-activated Cas12a make them impotent by disrupting the higher-ordered structure. After comparing four kinds of G-quadruplexspecific ligands, we selected Thioflavin T (ThT) to achieve the visual observation goal. The application feasibility was confirmed by combining with loop-mediated isothermal amplification (LAMP) for detection of Vibrio parahaemolyticus (V. parahaemolyticus), and it demonstrated a high sensitivity (1.36 x 10(2) copies) and specificity (among 12 kinds of bacteria). The method displayed similar performance as the real-time PCR for detection of real samples yet had lower requirement on the instrument. The signal output format could also distinguish Salmo salar from other seven kinds of sequence-similar Salmonidae. The findings and strategy proposed in this manuscript will expand the application of CRISPR/Cas12a-based label-free fluorescent analysis.

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