Zhou Ping-Ping, Zhang Jian-Zhong, You Yuan-Hai, Wu Yong-Ning
National Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, China.
Biomed Environ Sci. 2008 Feb;21(1):53-62. doi: 10.1016/S0895-3988(08)60007-0.
To develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray.
Two sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1).
A combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity.
A combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.
通过结合多重PCR和低密度DNA微阵列技术，开发一种同时检测抗草甘膦转基因大豆中多种靶基因的方法。
使用两组多重PCR系统扩增转基因大豆中的靶基因。根据多种已鉴定转基因作物中抗草甘膦大豆（GTS40-3-2）、玉米（Mon810、Nk603、GA21）、油菜（T45、MS1/RF1）和水稻（SCK）的遗传特性，设计了17种捕获探针（PCR产物）和17对相应引物。所有探针均被分类并鉴定为物种特异性探针。使用一个阴性探针和一个阳性对照探针评估所有反应的效率，从而消除任何假阳性和假阴性结果。多重PCR反应后，扩增产物用Cy5-dUTP掺假并与DNA微阵列杂交。然后扫描阵列以显示靶基因的特异性杂交信号。该方法应用于认证转基因大豆（抗草甘膦GTS40-3-2）和油菜（MS1/RF1）样品的分析。
成功开发了一种多重PCR和DNA微阵列相结合的技术，用于鉴定抗草甘膦大豆和MS1/RF1油菜中的多靶基因，具有很高的特异性和可靠性。在0.5%的转基因事件水平上实现了从转基因作物中可靠鉴定抗草甘膦转基因大豆的遗传特性，表明灵敏度很高。
多重PCR和低密度DNA微阵列相结合的技术能够可靠地检测和鉴定转基因作物。
