转基因大豆SHZD32-1转化体普通PCR和qRT-PCR检测方法的研究

摘要

Genetically modified (GM) soybean (Glycine Max) event SHZD32-1 was developed by Chinese scientists with independent intellectual property rights,and the event has herbicide resistance by translating glyphosate resistant gene G10-EPSPS into cultivated variety 'Zhongdou 32 (Glycine Max)'.At present,GM soybean event SHZD32-1 has entered the stage of environmental testing and has a broad application prospect.So far,there are little reports about detection methods for GM soybean event SHZD32-1.In order to protect and improve the implementation of laws and regulations for GM soybean event SHZD32-1,the specific primer pairs and probes were designed based on the flanking sequences between the soybean genome and inserted exogenous fragment of SHZD32-1.Conventional PCR and qRT-PCR detection methods had been developed in this study.Specificity detection of conventional PCR and qRT-PCR found that all mixture GM samples DNA could not obtain the positive results except containing the SHZD32-1 genomic DNA,indicating the methods were good for specificity.Conventional PCR could detect 0.1％ of GM SHZD32-1 event,and limit of detection (LOD) of qRT-PCR method could reach 21 copies of GM SHZD32-1 genomic DNA.The results were obtained by using the conventional PCR and qRT-PCR method for 60 repetitions under the LOD concentration.Therefore,the two methods had high specificity,good sensitivity,and strong stability.This research established GM SHZD32-1 specific PCR detection method is an important content of molecular characteristics of the evaluation of GMO,could be used to GMO safety regulation and provide technical support in China.%转基因大豆(Glycine max) SHZD32-1是我国自主研发的耐除草剂大豆转基因品系,该材料将耐草甘膦基因G10-EPSPS导入栽培品种中豆32 (Glycine Max,Zhongdou 32)使其获得耐除草剂抗性,具有广阔的应用前景.到目前为止,尚没有针对SHZD32-1的检测方法系统研究的报道.本研究以转基因大豆SHZD32-1转化体的插入位点基因组序列和外源插入片段/基因组连接区序列为靶标,通过设计和筛选引物、探针,特异性、灵敏度等测试,建立了特异性SHZD32-1转化体普通PCR和qRT-PCR检测方法.检测方法特异性实验中,普通PCR和qRT-PCR方法均只在含有SHZD32-1的样品中扩增出阳性结果,而其他不含有SHZD32-1的检测样品中均为阴性结果;方法灵敏性检测中,普通PCR检测法检出限为0.1％,qRT-PCR法检测下限(limit of detection,LOD)为2 1个拷贝;方法稳定性实验中,普通PCR和qRT-PCR方法在其相应最低检出限浓度下进行60次重复试验,结果均为阳性.本研究建立的SHZD32-1转化体特异性PCR检测方法特异性好、灵敏度高、稳定性强.转基因作物转化体特异性检测方法的研究是转基因生物分子特征评价的重要内容之一,其为转基因生物安全监管和追溯提供了技术支持.