基于Taqman-MGB多重实时荧光PCR快速鉴定抗除草剂转基因大豆品系  

Rapid Identification of Herbicide Resistant Transgenic Soybean Strains Based on Taqman-MGB Multiple Real-Time Fluorescence PCR

作　　者：王凤军 王美蓉 郑如福 陈锦辉 Wang Fengjun;Wang Meirong;Zheng Rufu;Chen Jinhui(Zhejiang Institute of Economic and Trade,Hangzhou 310012)

机构地区：[1]浙江经贸职业技术学院,杭州310012

出　　处：《中国粮油学报》2025年第1期211-219,共9页Journal of the Chinese Cereals and Oils Association

基　　金：浙江经贸职业技术学院省属高校基本科研业务费项目(24SBYB04)。

摘　　要：通过NCBI比对DAS-68416-4、DAS-44406-6、DAS-81419-2、SOYA 305423转基因大豆的序列同源性,在种属特异性区域设计多重引物和特异性荧光探针,同时提取基因组DNA,对退火温度和引物探针浓度进行优化,确定最佳反应程序,建立同时检测4种转基因大豆的多重实时荧光PCR方法。并对建立的实时荧光PCR反应进行特异性,重复性、灵敏性和适用性等方法学进行验证。结果表明DAS-68416-4、DAS-44406-6、DAS-81419-2、DP305423靶标序列的线性回归方程分别为y=-3.1524x+36.958,y=-3.1689x+38.828,y=-3.469x+37.933和y=-3.4839x+39.644;相关系数R 2分别为0.994、0.997、0.995和0.992;扩增效率分别为107%、106%、94.2%和93.7%,检出限可达9个拷贝,方法特异性强,重现性好,灵敏度高,是一种快速、经济、实用的检测方法,可对转基因大豆品系及其转化体成分进行大批量快速鉴定。The sequence homology of DAS-68416-4,DAS-44406-6,DAS-81419-2,and SOYA 305423 transgenic soybeans were compared by using NCBI,multiple primers and species-specific fluorescent probes were designed in specific regions and genomic DNA was extracted at the same time.The annealing temperature and primer probe concentration were optimized to determine the best reaction procedure,and a multiplex real-time fluorescent PCR method for simultaneous detection of four transgenic soybeans was established.And the methodology of specificity,repeatability,sensitivity,and applicability of the established real-time fluorescence PCR reaction was validated.The results indicated that the linear regression equations for the four target sequences of DAS-68416-4,DAS-44406-6,DAS-81419-2,and DP305423 were y=-3.1524x+36.958,y=-3.1689x+38.828,y=-3.469x+37.933,and y=-3.4839x+39.644,respectively;the correlation coefficients R 2 were 0.994,0.997,0.995,and 0.992,respectively;the amplification efficiency was 107%,106%,94.2%,and 93.7%,respectively,with a detection limit of up to 9 copies.The method had strong specificity,good reproducibility,and high sensitivity.It was a fast,economical,and practical detection method that could quickly identify genetically modified soybean strains and their transformed components into large quantities.

关 键 词：转基因大豆 品系鉴定 多重 实时荧光PCR 

分 类 号：S529[农业科学—作物学]