中文摘要：多靶标质粒DNA参考物质已被研发并被用来作为转基因生物分析中矩阵参考物质的良好替代品。本文构建了一个新型多靶标质粒参考分子pCAN，其含有八种转基因油菜品种（RF1、RF2、MS1、MS8、Topas 19/2、Oxy235、RT73、T45）的事件特异性序列和油菜内源参照基因PEP的部分序列。该质粒参考物质可用于八种转基因油菜品种的定性和定量PCR分析，包括特异性分析、检测限（LOD）、定量限（LOQ）、pCAN在各种油菜样品中分析的性能等。在定量实时PCR检测中，所有事件特异性和PEP检测的PCR效率均在91%-97%之间，且回归系数的平方均高于0.99。在模拟样品的定量分析中，定量偏差值在0.47%-20.68%之间，相对标准偏差（RSD）在1.06%-24.61%。研究结果表明，新型多靶标质粒参考分子pCAN在八种转基因油菜品种的检测和量化等常规分析中可作为质粒DNA参考物质的有效替代品。
外文摘要：Multiple-target plasmid DNA reference materials have been generated and utilized as good substitutes of matrix based reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid reference molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous reference gene PEP. The applicability of this plasmid reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), 'and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and PEP assays are between 91% and 97%, and the squared regression coefficients (R-2) are all higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of simulated samples. Furthermore, 10 practical canola samples sampled from imported shipments in the port of Shanghai, China, were analyzed employing pCAN as the calibrator, and the results were comparable with those assays using commercial certified materials as the calibrator. Concluding from these results, we believe that this newly developed pCAN plasmid is one good candidate for being a plasmid DNA reference material in the detection and quantification of the eight GM canola events in routine analysis.
外文关键词：multiple-target reference plasmid; genetically modified canola; qualitative PCR; quantitative real-time PCR
作者：Li, Zhuqing; Li, Xiang; Wang, Canhua; 等.
作者单位：Shanghai Jiao Tong Univ
期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
期刊影响因子：3.154
出版年份：2017
出版刊次：9
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