转基因玉米转化体特异性寡核苷酸芯片测试方法的研制

根据7种转基因玉米Bt11、Bt176、Mon810、Mon863、TC1507、GA21和NK603的重组DNA结构，利用Primer premier 5.0引物设计软件分别设计转化体特异性引物，优化PCR反应条件，对引物特异性及灵敏度进行验证。结果表明，所设计引物特异性强，灵敏度达0.1%。在植物基因组与外源基因的连接区域，利用Primer premier 5.0和Oligo 6.0软件分别设计和筛选7种转基因玉米40mer oligo转化体特异性探针，制备转基因玉米的寡核苷酸芯片，并对芯片检测的特异性、重复性及检测灵敏度进行检验。证明所设计探针特异性强，灵敏度达0.01%。与PCR检测方法相比，基因芯片检测法灵敏度高、特异性强，可有效检测及鉴定多种转基因玉米，大大提高检测的准确率和效率。

Developing a Method of Oiigonucleotide Microarray for Event Specific Detection of Transgenic Maize (Zea mays)

With the spread of genetically modified organism (GMO), the food and environment securities related to GMO attract great attention to the public. Genetically modified maize (Zea mays L.) is one of the most popular GMO varieties, which accounts for 22.5% of total planting area of transgenic crops. Currently, more than 40 countries and regions govern GMO food products with a compulsory tagging policy. Thus, technique for testing transgenic plants is of great importance, such as screening, gene specific, construct specific, event specific detection method, which have become a mainstay of GMOs detection. Event specific microarray shows a prosperous application due to its advantages of its high specificity, high sensitivity, automation and high efficiency. In this study, on the base of integrated DNA constructs of seven genetically modified maize varieties, Bt11, Bt176, Mon810, Mon863, TC1507, GA21, and NK603, event specific primers were designed for polymerase chain reaction (PCR) using Primer premier 5.0, and the PCR system and program were optimized. The results indicated that the sensitivity of PCR detection was 0.1%. In addition, based on unique and specific integration junction sequences between the host plant genome DNA and the integrated gene, a 40mer event specific oligo probe with high specificity was designed for each variety using Primer premier 5.0 and Oligo 6.0, all the event specific probes were developed in one microarray. To ensure the reliability of microarray detection, we used different kinds of positive and negative controls in oligonucleotide microarray. The results indicate that the sensitivity of microarray detection (0.01%) and the event specific oligonucleotide probes developed in this study meet the requirements of sensitivity and specificity for detecting and identifying genetically modified maize, which enhances the testing accuracy and efficiency.