利用可视化膜芯片检测9种转基因玉米  

Visual Membrane Chip for the Simultaneous Detection of Nine Genetically Modified Maizes

作　　者：李永进[1,2] 熊涛[2] 吴华伟[2] 杨亚珍[2] 

机构地区：[1]湖州师范学院生命科学学院,浙江湖州313000 [2]长江大学生命科学学院,湖北荆州434025

出　　处：《湖北农业科学》2016年第11期2926-2929,共4页Hubei Agricultural Sciences

基　　金：湖北省教育厅重点项目(D20141306)

摘　　要：建立了一种可同时检测9种转基因玉米(Zea mays L.)的可视化膜芯片检测方法。该方法利用紫外交联技术,将9种转基因玉米特异性探针及内源性玉米醇溶蛋白基因(Zein)探针和杂交质控探针固定于尼龙膜表面制成检测芯片。通过多重PCR同时扩增多重靶标片段,生物素化产物与检测芯片杂交。阳性结果由链酶亲和素-碱性磷酸酶及显色底物NBT/BCIP的显色反应在芯片表面形成裸眼可见的点。应用商业化转基因玉米(Bt176、Bt11、MON810、GA21、T25、TC1507、DAS-59122、NK603、MIR604)、转基因棉花(MON1445、MON15985)、转基因大豆及非转基因材料检验了芯片的性能,结果表明,制备的检测芯片具有良好的特异性和重复性,检测限为0.5%。芯片鉴定结果与PCR产物测序结果一致。A visual membrane chip was developed to simultaneously detect nine genetically modified maizes（Zea mays L.）. The chip comprised short oligonucleotide probes complimentary to the specific gene region for nine different genetically modified maizes（GMM）,endogenic zein gene probe and hybridization control probes. The multiplex PCR products annealed to the probe were reacted with streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium / 5-bromo-4-chloro-3 ＇-indolylphosphate,p-toluidine salt（NBT / BCIP）,resulting in blue spots that are easily visualized by unaided eyes for qualitative analysis.Commercial genetically modified maizes（Bt176,Bt11,MON810,GA21,T25,TC1507,DAS-59122,NK603,MIR604）,GM cotton（MON1445, MON15985）,GM soybean（monsanto roundup ready soybean 40-3-2） and non-GM materials were identified by this method and further confirmed by PCR plus sequencing. The results showed that each probe consistently identified its corresponding GMM target with a high specificity and good reproducibility. The limit of detection for GMM was 0.5%.

关 键 词：玉米(Zea mays L.) 可视化检测 膜芯片 转基因作物 

分 类 号：S513[农业科学—作物学]