摘要
旨在建立一套准确、快速、可靠的用于转基因大豆检测及DNA提取方法;以35S启动子(Cauliflowermosaicvirus 35S,CaMV 35S)、Nos终止子(Nopalinesynthase,Nos)、NPTⅡ、CP4-EPSPS四种外源基因和大豆内源基因(Lectin)为检测的目的片段,建立五重PCR反应体系,并采用改进的方法提取DNA,进行PCR扩增,实际检测了已知转基因大豆和市售大豆;改进的DNA提取方法与经典的CTAB方法相比,提取时间至少缩短了1h,降低了提取成本。经过PCR扩增对五种外源基因进行了检测,经DNA测序证实了产物为目的扩增产物。转基因大豆的检出限为0.2%～0.5%。改进后的DNA提取方法能够快速提取用于PCR扩增的高质量模板,消除了PCR反应的抑制因子;建立的五重PCR反应体系能够高效、准确的检测转基因大豆。
The aim of this work is to develop an accurate, rapid and reliable method for detection of genetically modified soybeans and extraction of DNA from genetically modified soybeans. Quintic PCR was successfully established with targeted genes including CaMV35S promoter, Nos tenninato, NPT Ⅱ , CP4 - EPSPS, and endogenous gene Lectin. DNA was isolated, by the improved method and then amplified by PCR. Genetically modified soybeans and commercial soybeans were detected with the quintic PCR. Results show that the improved CTAB method isolates total DNA quickly and reduces DNA - extraction cost as compared with the traditional CTAB method. Five different gene fragments amplified by PCR are identiffed with sequencing. The detection limit of the multiplex PCR is 0.2 % - 0.5 % for genetically modified soybeans. It is concluded that the improved method of DNA - extraction is rapid and accurate to extract high quality total DNA amplified by PCR and it eliminates the PCR inhibitor. The five - plex PCR reaction system is a highly effective and accurate way of detecting the genetically modified soybeans.
出处
《中国粮油学报》
EI
CAS
CSCD
北大核心
2008年第3期194-198,共5页
Journal of the Chinese Cereals and Oils Association
基金
河北农业大学科技发展基金(2005-01)
关键词
转基因
大豆
DNA提取
多重PCR
检测
transgenic, soybean, DNA extraction, multiplex PCR, detection
