Development of an event-specific Real-time PCR detection method for the transgenic Bt rice line KMD1
Development of an event-specific Real-time PCR detection method for the transgenic Bt rice line KMD1
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DOI：
10.1007/s00217-008-0981-0
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发表时间：
2009-03-01
影响因子：
3.3
通讯作者：
Busch, Ulrich
中科院分区：
文献类型：
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作者：
Babekova, Ruzha;Funk, Tristan;Busch, Ulrich
本研究建立了转Bt基因水稻科明稻1号（KMD 1）的事件特异性荧光定量PCR检测方法。这一尚未在任何国家获得批准的水稻品系很可能在不久的将来在中国获得批准。开发的引物扩增跨越KMD 1中遗传构建体的整合位点的DNA序列。使用SiteFinding-PCR技术获得了引物设计所需的该未知位点的DNA序列信息。检测方法的特异性通过测试许多不同的转基因和常规植物品种（例如水稻LL 601、LL 62、Bt 63）来显示。作为基因组DNA的替代，我们开发了双靶杂交扩增子作为实时PCR中的合成校准标准。这些扩增子含有KMD 1事件特异性序列的一个拷贝和水稻参考基因gos 9序列的一个拷贝。该方法的定量限（LOQ）为0.05%。
The present study describes an event-specific quantitative Real-time PCR detection method for the transgenic Bt rice line Kemingdao 1 (KMD1). This rice line which is not approved in any country so far is likely to be approved in China in the near future. The developed primers amplify a DNA sequence spanning the integration site of the genetic construct in KMD1. DNA sequence information of this unknown site necessary for primer design was achieved using SiteFinding-PCR technique. The specificity of the detection method was shown by testing a number of different transgenic and conventional plant varieties (e.g. rice LL 601, LL 62, Bt 63). As alternative to genomic DNA, we developed double target hybrid amplicons as synthetic calibration standards in Real-time PCR. These amplicons contained both one copy of the KMD1 event-specific sequence and one copy of a sequence of the rice reference gene gos9. The limit of quantification (LOQ) of the method was tested to be 0.05%.