抗除草剂棉花LLCOTTON25的多重PCR检测方法

为了建立1种抗除草剂棉花LLCOTTON25多重PCR检测方法,根据抗除草剂棉花LLCOTTON25分子特征,同时选择棉花内源参照基因(SADI)、花椰菜花叶病毒启动子(P-Ca MV 35S)、根癌农杆菌终止子(T-NOS)和目的基因(bar)4个基因作为多重PCR(Polymerase chain reaction)检测基因,参照国家相关标准中的特异性引物序列,通过对反应条件的优化以及方法特异性和灵敏度测试,建立了可同时检测除内源基因外的3种外源目的基因的多重PCR检测体系。利用已知样品对本体系验证,抗除草剂棉花LLCOTTON25能被同时检出SADI、P-Ca MV 35S、T-NOS、bar等4个基因,而其他样品均不能被同时检出这4个基因。结果表明此体系可运用于抗除草剂棉花LLCOTTON25检测。

Multiplex PCR for the Detection of Herbicide-Resistant Cotton,LLCotton25

This study aimed to establish a multiplex PCR detection method for the herbicide-resistant cotton line, LLCotton25. We selected the endogenous cotton reference gene SADI, the cauliflower mosaic virus promoter(P-CaMV 35S), Agrobacterium tumefaciens terminator(T-NOS), and the target gene(bar) as detection genes for multiplex PCR.We optimized PCR reaction conditions using sequences of specific Chinese national standard primers, and tested its specificity and sensitivity. The multiplex PCR detection system was then used to successfully detect the four genes SADI, P-CaMV 35S, T-NOS, and bar from the herbicide-resistant cotton LLCotton25, but not from other samples. These results indicate that this system is sufficiently specific to detect the herbicide-resistant cotton LLCotton25.