转基因水稻克螟稻2号定量检测用质粒标准分子研制及不确定度评价  

Construction of a Reference Plasmid for the Quantification of Genetically Modified Rice Kemingdao 2 and Uncertainty Evaluation

作　　者：李亮[1] 臧超[1] 王晶[1] 隋志伟[1] 赵正宜[1] 董莲华[1] 

机构地区：[1]中国计量科学研究院,北京100013

出　　处：《中国生物工程杂志》2012年第10期19-24,共6页China Biotechnology

基　　金：国家科技支撑计划资助项目(2008BAK41B01)

摘　　要：转基因作物的食用安全和环境安全一直受到消费者与各国政府及相关机构人员高度重视。质粒标准分子是转基因核酸量值的载体,为实现转基因植物核酸量值准确性、可比性和有效性提供保障。描述了转基因水稻克螟稻2号质粒标准分子(pKMD2)的研制过程,包括质粒构建、特异性、均匀性、稳定性、可替代性和量值及不确定度评价等方面。量值结果表明pKMD2质粒标准分子所含两个基因片段比值为1.032,不确定度为0.032,可以替代基因组作为阳性标准品用于实验室质控、含量检测及贸易争端等领域。With the rapid increase in the number of biotech crops used for food production,consumers,researchers and governments are concerned about the health risks and environment safety posed by genetically modified（GM） crops and their derivatives.Reference plasmids are carriers of GM nucleic acid of the value and are provided for the value accuracy,comparability and effectiveness of realization of GM nucleic acid.Here,a novel reference plasmid for quantification of Kemingdao2（KMD2） was reported.Plasmidic DNA containing rice KMD2 DNA fragments is used as real-time PCR standard and positive PCR control.The pKMD2 plasmid was constructed based on pEASY-T3 that includes a 106 bp fragment of RBE4 which enables positive control PCRs,and a 137 bp GM constructed specific fragment of KMD2 for foreign gene quantification.The preparation,homogeneity,stability and commutability of pKMD2 were processed.To verify the correct number ratio between the RBE4 and constructed specific fragments in the pKMD2,the data was generated from sequencing by three independent laboratories and performed by quantitative real-time PCR for batch characterization,resulted 1.032.The uncertainty was evaluated by the sources of homogeneity,stability and batch characterization,resulted 0.032.In conclusion,this newly constructed plasmid is suitable for the practical detection and quantification of GM rice Kemingdao2 and feed products containing this DNA fragment.

关 键 词：转基因水稻 质粒标准分子 不确定度 定量 

分 类 号：Q819[生物学—生物工程]