耐除草剂大豆'DBN9004'精准定量检测方法的建立  

Establishment of Accurate Quantitative Detection Method for Herbicidetolerant Soybean(Glycine max)'DBN9004'

作　　者：赵新[1] 刘双 刘娜[1] 李瑞环 高芳瑞 兰青阔[1] 梁晋刚 王永[1] ZHAO Xin;LIU Shuang;LIU Na;LI Rui-Huan;GAOFang-Rui;LAN Qing-Kuo;LIANG Jin-Gang;WANG Yong(Tianjin Academy of Agricultural Sciences,Tianjin 300384,China;Development Center of Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100176,China)

机构地区：[1]天津市农业科学院,天津300384 [2]农业农村部科技发展中心,北京100176

出　　处：《农业生物技术学报》2022年第12期2446-2455,共10页Journal of Agricultural Biotechnology

基　　金：农业农村部2020年农业国家标准和行业标准制修订项目(农质标函〔2020〕128号)。

摘　　要：耐除草剂大豆(Glycine max)'DBN9004'是我国自主研发的对除草剂草甘膦和草铵膦同时具有耐受性的转基因大豆新品种,已获得安全证书,在中国具有重要的产业化应用前景。为满足转基因作物安全监管技术要求,亟需对其进行定量检测方法的研发。本研究以耐除草剂大豆'DBN9004'的3'端旁侧序列为靶标,设计引物和探针,经特异性、正确度、精密度、检出限、定量限等指标测试,建立了该转化体特异性qPCR和微滴数字PCR检测方法。结果显示,本方法特异性强、重复性好,定量限达0.1%(约40个拷贝),检出限达0.05%(约20个拷贝),测试样品定值准确度高,相对标准偏差(RSD)为6.00%~10.95%,且qPCR与微滴数字PCR方法定量结果一致。该方法为耐除草剂大豆新品种'DBN9004'的精准定量检测提供了一种新的技术手段,为农业转基因监管提供技术支撑。Herbicide-tolerant soybean(Glycine)'DBN9004'is a new genetically modified soybean event with tolerance to both glyphosate and glufosinate.It has obtained safety certificate and has important industrial application prospect in China.In order to meet the technical requirements of safety supervision for genetically modified crops,it is urgent to develop quantitative detection methods for'DBN9004'.In this study,the 3'flanking sequence of herbicide-tolerant soybean'DBN9004'was used as the target,primers and probes were designed,and a qPCR assay was established.The specificity,accuracy,precision,detection limit,quantitative limit and other indicators were tested.The results showed that the method was specific and reproducible,with a quantitative limit of 0.1%(40 copies)and a detection limit of 0.05%(20 copies),the relative standard deviation(RSD)was 6.00%~10.95%,and the quantitative results of qPCR and droplet digital PCR method were consistent.This method provides a new technical means for the accurate quantitative detection of new herbicide-tolerant soybean event'DBN9004',and provides technical support for the regulation of agricultural transgene crops.

关 键 词：耐除草剂大豆 'DBN9004' qPCR 微滴数字PCR 精准定量 

分 类 号：S565.1[农业科学—作物学]