State Key Laboratory for Quality and Safety of Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China; Key Laboratory of Information Traceability for Agricultural Products, Ministry of Agriculture of China, Hangzhou, 310021, China.
State Key Laboratory for Quality and Safety of Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China; College of Chemistry and Life Science, Shenyang Normal University, Shenyang, 110034, China.
Anal Chim Acta. 2020 May 1;1109:158-168. doi: 10.1016/j.aca.2020.02.044. Epub 2020 Feb 25.
With the large-scale planting of genetically modified (GM) crops, the development of a rapid and convenient method for on-site monitoring GM crops is needed. In this study, a duplex recombinase polymerase amplification (DRPA)-based, quick and simple detection system is presented for on-site detection of GM crops. In this system, a rapid DNA extraction method, a DRPA, and a lateral flow biosensor (LFB) are integrated to allow for rapid DNA extraction and amplification, and fast visualization of the detection results. Using the rapid DNA extraction method, high-quality DNA suitable for RPA and polymerase chain reaction (PCR) was quickly isolated from various crops within 5 min. Utilizing the optimal DRPA assay, the universal screening sequences (Cauliflower mosaic virus 35S promoter [35S] and Agrobacterium tumefaciens NOS terminator [NOS]) were rapidly and simultaneously amplified with high selectivity and sensitivity. The sensitivity threshold of the DRPA assay was ∼10 copies for GM soybean genomic DNA and 100 ng DNA of 0.1% GM soybean. In combination with the LFB in an enclosed cassette, the entire detection process was performed in approximately 20-30 min and eliminated the carryover contamination. No special or expensive equipment was needed for the detection process. The system was successfully applied and validated for on-site detection of GM rice, demonstrating its suitability for on-site testing of GM crops and high potential for application to other fields, especially in low-resource regions that require rapid detection of DNA targets.
随着转基因（GM）作物的大规模种植，需要开发一种快速、便捷的现场监测 GM 作物的方法。本研究提出了一种基于双重组酶聚合酶扩增（DRPA）的快速简便的检测系统，用于现场检测 GM 作物。在该系统中，集成了快速 DNA 提取方法、DRPA 和横向流动生物传感器（LFB），以实现快速 DNA 提取和扩增，并快速可视化检测结果。通过快速 DNA 提取方法，在 5 分钟内即可从各种作物中快速分离出适合 RPA 和聚合酶链反应（PCR）的高质量 DNA。利用最佳的 DRPA 分析，通用筛选序列（花椰菜花叶病毒 35S 启动子[35S]和根癌农杆菌 NOS 终止子[NOS]）可以快速且同时进行高选择性和高灵敏度的扩增。DRPA 分析的灵敏度阈值约为 10 个拷贝的 GM 大豆基因组 DNA 和 0.1% GM 大豆的 100ng DNA。与封闭盒中的 LFB 结合使用，整个检测过程大约需要 20-30 分钟，并且消除了交叉污染。检测过程不需要特殊或昂贵的设备。该系统成功应用于现场检测 GM 水稻，并验证了其适用于 GM 作物的现场检测，并且具有在其他领域应用的高潜力，特别是在需要快速检测 DNA 靶标的资源匮乏地区。
