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以A2704-12、A5547-127、MON89788和GTS-40-3-2等4种商品化耐除草剂转基因大豆为材料,构建质粒标准分子用于解决转基因作物检测时标准物质缺乏现状。采用双酶切技术,将扩增的大豆内参基因和耐除草剂转基因大豆转化体特异性片段克隆至pMD18-T载体上。结果显示,质粒标准分子定性PCR特异性序列片段的LOD均为20copies/μL,定量检测体系Bias最大值≤9.89%,CV最大值≤5.96%,SD最大值≤0.06,实验得到的所有偏差值均在允许范围之内,构建的质粒标准分子适用于4种耐除草剂转基因大豆特异性检测。
Four commercial herbicide-tolerant genetically modified soybeans, A2704-12, A5547-127, MON89788 and GTS-40-3-2, were used as materials to construct plasmid standard molecules for the purpose of solving the lack of standard substances in transgenic crops. Double-digestion technique was used to clone the amplified soybean internal reference gene and the herbicide tolerant transgenic soybean transformant specific fragment into pMD18-T vector. The results showed that the LOD of qualitative PCR-specific fragment of plasmid standard molecule was 20copies / μL, the maximum value of Bias in quantitative detection system was less than 9.89%, the maximum value of CV was ≤ 5.96% and the maximum value of SD was ≤0.06. All the deviation values Within the allowable range, the constructed plasmid standard molecules are suitable for the specific detection of four herbicide resistant transgenic soybeans.