转基因大豆、玉米、油菜和水稻品系特异性检测质粒标准分子的快速构建及应用  

Construction and Application of Standard Reference Molecule for Strain-specific Detection in Genetically Modified Soybean,Maize,Rape and Rice

作　　者：吴永彬[1] 肖维威[1] 张宝[1] 蔡翠霞[1] 康文杰[1] 周琳华[1] 刘唐书 马文丽[1] 

机构地区：[1]南方医科大学基因工程研究所,广州510515 [2]广东省产品质量监督检验研究院,广州510330

出　　处：《中国生物化学与分子生物学报》2011年第8期775-782,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家公益性行业科研专项(No.200910265);广东省科技计划项目(No.2008B080701027)~~

摘　　要：本文采用重叠延伸PCR技术快速构建了转基因大豆GTS40-3-2、玉米NK603、油菜RT73和水稻TT51-1的4种品系作物的质粒标准分子.经快速PCR鉴定及测序分析验证后,将构建的阳性质粒标准分子应用于实时荧光定量PCR标准曲线的构建,并建立其相应的荧光定量PCR检测体系,同时对该体系的扩增效率、精确度、灵敏度等指标进行了评估.结果显示,建立的实时荧光定量PCR检测体系中,目标序列的扩增效率均在97.434%～101.479%正常范围内(R2≥0.995),定量极限为20 copies,表明我们已成功构建了这4种转基因作物的品系质粒标准分子,并能有效应用于实时荧光定量PCR标准曲线的构建.Using the overlap extension PCR method,four reference plasmids of genetically modified（GM） soybean GTS40-3-2,GM maize NK603,GM rape RT73,GM rice TT51-1 stains were constructed.After verified by PCR and sequencing,the standard curves were obtained for the quantification of the reference molecules by real-time quantitative PCR.The results showed that the PCR efficiencies of the target sequence detection were within a normal range of 97.434~101.479%（R2≥0.995）,and the limits were as low as 20 copies.These indicated that the standard reference molecules for the positive detection in the four GM crop stains were successfully validated and could be used as the reference materials（RM）.

关 键 词：转基因 重叠延伸PCR 质粒标准分子 品系特异性检测 实时荧光定量PCR 

分 类 号：TS207.3[轻工技术与工程—食品科学]