Development of a novel Cas13a/Cas12a-mediated 'one-pot' dual detection assay for genetically modified crops

文献类型： 外文期刊

第一作者： Ding, Lin

作者： Ding, Lin;Wang, Xiaofu;Chen, Xiaoyun;Xu, Xiaoli;Wei, Wei;Yang, Lei;Ji, Yi;Xu, Junfeng;Peng, Cheng;Wu, Jian

作者机构：

关键词： Dual CRISPR; Genetically Modified (GM) detection; One-pot reaction; Recombinase polymerase amplification; (RPA); Multiplexed lateral flow strips

期刊名称：JOURNAL OF ADVANCED RESEARCH （ 影响因子：13.0； 五年影响因子：11.6 ）

ISSN： 2090-1232

年卷期： 2025 年 72 卷

页码：

收录情况： SCI

摘要： Introduction: Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation. Objective: In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection. Methods: The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically. Results: Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability. Conclusion: The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments. (c) 2024 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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