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A Localized CRISPR Assay that Detects Short Nucleic Acid Fragments in Unamplified Genetically Modified Samples

文献类型： 外文期刊

作者： Peng, Cheng 1 ; Wang, Yuling 2 ; Chen, Xiaoyun 1 ; Wang, Xiaofu 1 ; Ding, Lin 1 ; Xu, Xiaoli 1 ; Wei, Wei 1 ; Yang, Lei 1 ; Wu, Jian 3 ; Sun, Meihao 2 ; Xu, Junfeng 1 ;

作者机构： 1.Zhejiang Acad Agr Sci, State Key Lab Managing Biot & Chem Threats Qual &, Key Lab Traceabil Agr Genet Modified Organisms, Minist Agr & Rural Affairs, Hangzhou 310021, Peoples R China

2.Zhejiang Normal Univ, Coll Chem & Life Sci, Jinhua 321004, Peoples R China

3.Zhejiang Univ, Coll Biosyst Engn & Food Sci, Hangzhou 310058, Peoples R China

关键词： CRISPR (clustered regularly interspaced short palindromic repeats); genetically modified organism detection; amplification-free; short nucleic acid fragments; dual-quenching system

期刊名称：ACS SENSORS （ 影响因子：8.9； 五年影响因子：9.0 ）

ISSN： 2379-3694

年卷期： 2023 年 8 卷 3 期

页码：

收录情况： SCI

摘要： Detecting short genetically modified (GM) nucleic acid fragments in GM crops and associated products is critically important for the global agriculture industry. Although nucleic acid amplification-based technologies have been widely used for genetically modified organism (GMO) detection, they still struggle to amplify and detect these ultra-short nucleic acid fragments in highly processed products. Here, we used a multiple-CRISPR-derived RNA (crRNA) strategy to detect ultra-short nucleic acid fragments. By combining confinement effects on local concentrations, an amplification-free CRISPR-based short nucleic acid (CRISPRsna) system was established to detect the cauliflower mosaic virus 35S promoter in GM samples. Moreover, we demonstrated assay sensitivity, specificity, and reliability by directly detecting nucleic acid samples from GM crops with a wide genomic range. The CRISPRsna assay avoided possible aerosol contamination from nucleic acid amplification and saved time due to an amplification-free approach. Given that our assay displayed distinct advantages over other technologies in detecting ultra-short nucleic acid fragments, it may have wide applications for detecting GM in highly processed products.

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