Integrated structure and event-specific real-time detection of transgenic cry1Ac/SCK rice Kefeng 6

文献类型： 外文期刊

第一作者： Su, Changqing

作者： Su, Changqing;Xie, Jiajian;Wang, Xifeng;Peng, Yufa;Su, Changqing

作者机构：

关键词： Cry1Ac/SCK rice;Kefeng 6;Integration junction;Event-specific PCR;Reference molecule;Real-time PCR

期刊名称：EUROPEAN FOOD RESEARCH AND TECHNOLOGY （ 影响因子：2.998； 五年影响因子：3.005 ）

ISSN： 1438-2377

年卷期： 2011 年 232 卷 2 期

页码：

收录情况： SCI

摘要： Transgenic rice Kefeng 6 is a transformation event containing two insect-resistant genes, cry1Ac and SCK (modified CpTI gene) in China. In order to monitor the probable release of Kefeng 6 in the future and execute the labeling requirements, it is necessary to develop a rapid and reliable detection method. In this study, both the 5' and 3'-junction sequences spanning the plant DNA and the integrated gene construct of the rice event Kefeng 6 were isolated by genome walking and long-distance PCR (LD-PCR), successively. Multiple copies of truncated SCK gene and cry1Ac gene were found to integrate into the host rice genome. The event-specific real-time detection method for Kefeng 6 event based on its 5'-junction sequence was established using one plasmid molecule pMD-KF6 containing both 5'-junction sequence and rice endogenous gene gos9 sequence as the reference material (RM) with an absolute limit of quantification (LOQa) around 10 template copies. Thereafter, three different transgenic amounts of w/w mixed samples (5, 1, and 0.5%, respectively) were quantified to assess the performance characteristics of the established real-time PCR method. The accuracy expressed as bias deviated from the 4.00-26.00%, the precision expressed as standard deviation (SD) and relative standard deviation (RSD) deviated from 0.03-0.19 and 3.42-4.76%, respectively. Based on the earlier results, we concluded that the qualitative and quantitative PCR assays were reliable and accurate for Kefeng 6 measurement, and the reference plasmid pMD-KF6 could be a good substitute for the reference material for Kefeng 6 quantification.

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