摘要
转化体标准化检测方法为生物育种产业化的应用和定量标识制度实施提供了重要的技术支撑。基于插入序列左边界的信息,以耐除草剂大豆LP012-1转化体为研究对象,建立了实时荧光定量PCR(quantitative real-time PCR,qPCR)检测方法。以外源插入序列位点左边界为靶标序列设计引物及其相应的探针,筛选获得了最优的引物组合,并优化引物探针浓度和退火温度,确定线性动力学范围在0.018~100 ng,检出限和定量限分别为0.05%、0.10%;正确度、精密度、实验室内稳健性和实验室间的联合验证误差均小于25%。研究建立的qPCR检测方法特异性好、灵敏度高、稳健性强,可以为耐除草剂转基因大豆LP012-1的安全监管提供技术支撑,并服务于生物育种商业化。
The standardized genetically modified detection′s method provides important technical support for the industrial application and quantitative identification system implementation of biological breeding.Based on the left boundary of the foreign insertion sequence,a quantitative real-time PCR(qPCR)detection method was established with the genetically modified soybean LP012-1 with genetically modified herbicide-tolerant soyabean selected as the research object.The target sequence of the left boundary of the foreign insertion sequence was designed as the primer and its corresponding probe,and the optimal primer combination was selected by screening.The primer and probe concentrations and annealing temperature were optimized,and the linear dynamic range was determined to be from 0.018 to 100 ng.The detection limit and the quantitative limit were 0.05% and 0.10%,respectively.The correctness,precision,robustness,and joint validation error between laboratories were less than 25%.The qPCR detection method established in this study has good specificity,high sensitivity,and strong robustness,which can provide technical support for the safety supervision of transgenic soybean LP012-1 and serve the commercialization of biological breeding.
作者
张月秋
傅芳奇
张华
陈一帆
王晨尧
蒋红叶
李亚辉
廖一尘
王丹
孙宇
付伟
陈红
ZHANG Yueqiu;FU Fangqi;ZHANG Hua;CHEN Yifan;WANG Chenyao;JIANG Hongye;LI Yahui;LIAO Yichen;WANG Dan;SUN Yu;FU Wei;CHEN Hong(Development Center of Science and Technology,Ministry of Agriculture and Rural Affairs,Beijing 100176,China;Shanghai Academy of Agricultural Sciences,Shanghai 201199,China;Institute of Food Safety and Nutrition,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)
出处
《生物技术进展》
2025年第2期276-286,共11页
Current Biotechnology
基金
农业生物育种国家科技重大专项(2022ZD04020)。
