小麦内源基因GAG56D的快速检测技术体系

Rapid Detection of Endogenous Gene GAG56D in Wheat by LAMP

为了满足转基因小麦检测中对内源参照基因的检测要求,以小麦属特异的内源基因γ-醇溶蛋白基因(GAG56D)作为目的基因,采用环状等温扩增技术(LAMP)建立了小麦内源基因GAG56D的快速检测技术体系。针对小麦内源基因GAG56D特异靶序列的6个区域设计4条特异性引物,通过显色法进行LAMP检测,并对时间、温度等反应条件及反应灵敏度、特异性、稳定性进行探索。结果表明,该检测体系最适反应温度为63℃,反应时间60min,定性检测低限达到0.5%(w/w)。该检测方法具有高度的特异性、灵敏度和稳定性,操作简单、快速。

The objective of this paper is to establish a rapid detection method for the reference gene in wheat,which is a necessary step in the detection of genetically modified wheat, A loop mediated iso- thermal amplification （LAMP） method was developed in this article, targeting to 7- GAG55D , the specific endogenffous gene in wheat. Based on the LAMP method, four specific primers for wheat were designed according to the sequences in six target region of GAG56D , and the reactive conditions were optimized. At last, visual detection could confirm the results of LAMP amplification. The re- suits showed that the optimum temperature and reaction duration was 63℃ and 60 rain, respectively. The sensitivity of LAMP detection was 0.5% （w/w）. Results indicated that LAMP was a sensitive, specific, stable and quick method for the detection of GAGSCD in wheat.