RNAi转基因大豆品系‘B5C9123-5’的RPA可视化检测技术  

RPA visual detection technology for RNAi transgenic soybean‘B5C9123-5’

作　　者：张晨[1] 雷展 李凯 李飞武[4] 商颖[1] 许文涛 ZHANG Chen;LEI Zhan;LI Kai;LI Feiwu;SHANG Ying;XU Wentao(College of Agriculture and Food,Kunming University of Science and Technology,Kunming 650504,China;College of Nutrition and Health/Beijing Advanced Innovation Center for Food Nutrition and Human Health,China Agricultural University,Beijing 100083,China;Key Laboratory of Safety Assessment of Genetically Modified Organism(Food Safety),Ministry of Agriculture and Rural Affairs,Beijing 100083,China;Institute of Agricultural Quality Standard and Testing Technology,Jilin Academy of Agricultural Sciences,Changchun 130033,China)

机构地区：[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

出　　处：《中国农业大学学报》2022年第1期50-57,共8页Journal of China Agricultural University

基　　金：转基因重大专项(2018ZX0801102B-001)。

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20min内用肉眼鉴别,检测限为1.00ng/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。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‘B5 C9123-5’.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.

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 

分 类 号：D550.1099[政治法律—政治学]