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

被引:1
作者
Changqing Su
Jiajian Xie
Xifeng Wang
Yufa Peng
机构
[1] Chinese Academy of Agricultural Sciences,Inspection Test Center for Environmental Safety of Transgenic Crops (Beijing), Ministry of Agriculture, State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant Protection
[2] HengShui College,Department of Life Science
来源
European Food Research and Technology | 2011年 / 232卷
关键词
rice; Kefeng 6; Integration junction; Event-specific PCR; Reference molecule; Real-time PCR;
D O I
暂无
中图分类号
学科分类号
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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页码:351 / 359
页数:8
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