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| RNAi转基因大豆品系‘B5C9123-5’的RPA可视化检测技术 |
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张晨1,雷展1,李凯2,3,李飞武4,商颖1*,许文涛2,3*
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| (1.昆明理工大学 农业与食品学院, 昆明 650504;2.中国农业大学 食品科学与营养工程学院/北京食品营养与人类健康高精尖创新中心, 北京 100083;3.农业农村部农业转基因生物安全评价(食用)重点实验室, 北京 100083;4.吉林省农业科学院 农业质量标准与检测技术研究所, 长春 130033) |
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| 摘要: |
| 为开发RNAi转基因作物的田间可视化鉴定方法，以DNA嵌合染料SYBR Green I为材料，采用设置重组酶聚合酶扩增(Recombinase Polymerase Amplification，RPA)特异性引物的手段，建立了一种快速、低成本、可视化的RPA用于转基因大豆‘B5C9123-5’的检测。结果表明，在靶标不存在时，RPA体系中游离的SYBR Green I染料使溶液呈现较弱的黄色荧光。在靶标存在的情况下，RPA扩增产生的双链DNA与SYBR Green I结合导致溶液从黄色转变为绿色并使荧光迅速增强。通过对RPA扩增时间和温度进行优化，阳性结果可在20 min内用肉眼鉴别，检测限为1.00 ng/μL。通过设置不同RPA引物，该方法可用于现场快速检测多种RNAi转基因作物。 |
| 关键词: RNAi转基因作物 重组酶聚合酶扩增 可视化检测 SYBR Green I |
| DOI：10.11841/j.issn.1007-4333.2022.01.05 |
| 投稿时间：2021-02-03 |
| 基金项目:转基因重大专项(2018ZX0801102B-001) |
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| RPA visual detection technology for RNAi transgenic soybean ‘B5C9123-5' |
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ZHANG Chen1,LEI Zhan1,LI Kai2,3,LI Feiwu4,SHANG Ying1*,XU Wentao2,3*
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| (1.College of Agriculture and Food, Kunming University of Science and Technology, Kunming 650504, China;2.College of Nutrition and Health/Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing 100083, China;3.Key Laboratory of Safety Assessment of Genetically Modified Organism(Food Safety), Ministry of Agriculture and Rural Affairs, Beijing 100083, China;4.Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences, Changchun 130033, China) |
| Abstract: |
| In order to develop visual identification methods for RNAi transgenic crops in the field, a DNA chimeric dye SYBR Green I was used as material, and RPA-specific primers were set up. A fast, low-cost and visualized Recombinase Polymerase Amplification(RPA)technique was developed for the detection of transgenic soybean ‘B5C9123-5'. The results show that: SYBR Green I in the RPA system made the solution appear yellow and weakly fluorescent in the absent of the target. In the presence of the target, the double-stranded DNA produced by RPA amplification bound to SYBR Green I, which caused the color of solution changing from yellow to green and a rapid increase in fluorescence. By optimizing the amplification time and temperature of RPA, the positive results could be identified by naked eye within 20 min with a detection limit of 1. 00 ng/L. By setting different RPA primers, this method can be used to quickly detect a variety of RNAi transgenic crops in the field. |
| Key words: RNAi transgenic crops recombinase polymerase amplification visual detection SYBR Green I |
