Event-specific qualitative and quantitative PCR detection methods for Transgenic rapeseed hybrids MS1 x RF1 and MS1 x RF2

文献类型： 外文期刊

第一作者： Wu, Yuhua

作者： Wu, Yuhua;Wu, Gang;Xiao, Ling;Lu, Changming

作者机构：

关键词： transgenic rapeseed;event-specific detection;quantification PCR;gene-stacked hybrid;male sterile;fertility restorer

期刊名称：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY （ 影响因子：5.279； 五年影响因子：5.269 ）

ISSN： 0021-8561

年卷期： 2007 年 55 卷 21 期

页码：

收录情况： SCI

摘要： Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1 x RF1 and MS1 x RF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1 x RF1 and MS1 x RF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1 x RF1 and MS1 x RF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.

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