转基因大豆质粒DNA标准物质的研制  

Development of a Plasmid DNA Reference Material for PCR Analysis of Genetically Modified Roundup Ready Soybean

作　　者：梁文[1] 许丽[1] 李兰英[1] 李妍[1] 闻艳丽[1] 徐勤[1] 任淑贞[1] 刘刚[1] 

机构地区：[1]上海市计量测试技术研究院化学与电离辐射所,上海201203

出　　处：《实验室研究与探索》2016年第5期18-21,45,共5页Research and Exploration In Laboratory

基　　金：国家质检公益性行业科研专项(201310016);上海市计量测试技术研究院项目(HOORY1406)

摘　　要：为了方便、快速地检测大豆及其加工食品中的转基因成分,准确定量转基因大豆的转基因含量,构建含常用转基因插入元件花椰菜花叶病毒Ca MV35S启动子、NOS终止子、新霉素磷酸转移酶基因NPTⅡ、玄参花叶病毒FMV35S启动子和大豆内源基因Lectin-1的质粒DNA标准物质,质粒DNA中靶基因元件均为单拷贝。开展了质粒DNA标准物质的均匀性、稳定性、检测适用性、定值及不确定度评价。结果表明,在实时荧光定量PCR(q PCR)检测中,质粒DNA的4种转基因插入元件分别与大豆内源基因Lectin-1的比值为0.98±0.06(Ca MV35S:L-1),0.95±0.06(FMV35S:L-1),1.05±0.08(NOS:L-1),1.11±0.08(NPTII:L-1)(k=2),靶基因扩增的标准曲线R2>0.99,可用于转基因检测实验室的结果质量控制,能力验证等活动。In order to find a simplified and accurate detection of the genetically modified soybean and its processed food,we developed an unique plasmid molecule for genetically modified soybean PCR analysis,it contained four exogenous target DNA element of genetically modified organisms（ Ca MV35 S,NOS,NPTII and FMV35S）,and one element of soybean endogenous（ Lectin-1 gene,L-1）. All the target genes were inserted into the plasmid with single copy. We quantified the ratios of the exogenous target DNAs and the L-1 as the certified valued of the reference material,and we evaluated the uniformity,stability,and the uncertainty,and examined the applicability of our plasmid. The certified values and expanded uncertainties（ k = 2） for the candidate reference material in the real time PCR were 0. 98 ± 0. 06（ Ca MV35S： L-1）,0. 95 ± 0. 06（ FMV35S： L-1）,1. 05 ± 0. 08（ NOS： L-1）,1. 11 ± 0. 08（ NPTII： L-1）,respectively.The R2 values of the standard curve for target genes were all higher than 0. 99. The plasmid reference material can be used in quality control and proficiency testing for GMOs analysis. It is innovative that plasmid DNA standard material containing multiple transgenic testing element and the ratio of gene copy number for each element and soybean is 1∶ 1.

关 键 词：转基因大豆 质粒标准物质 不确定度评定 

分 类 号：Q819[生物学—生物工程]