抗除草剂棉花LLCOTTON25的多重PCR检测方法

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

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。

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 con- ditions 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 herbi- cide-resistant cotton LLCotton25, but not from other samples. These results indicate that this system is sufficiently specific to de- tect the herbicide-resistant cotton LLCotton25.