转基因水稻和转基因油菜多重PCR检测技术研究

发布时间：2018-04-08 17:00

  本文选题：多重PCR　切入点：转基因水稻　出处：《沈阳农业大学》2017年硕士论文

【摘要】：随着全球转基因作物及其产品的数量和种类越来越多,其所带来的潜在风险也备受人们的关注。转基因生物安全监管事关粮食安全、食品安全和生态安全,以欧盟为代表的国家和地区出台了一系列的法律法规,加强监管。转基因成分检测是转基因生物安全监管的重要技术支撑和手段。近年来,基于传统PCR检测技术已经不能满足转基因成分检测简便快捷高效日益发展的需求。多重PCR(Multiplex PCR)技术因特异性强、灵敏度高、成本低、通量高、节省实验样品和药品等诸多优点,已经在转基因成分检测研究领域得到了广泛的研究和应用。本文以转基因水稻TT51-1、KMD1、KF6和转基因油菜RF1、MS8、Topas19/2、Oxy235、T45、GT73、RF2、RF3等为研究对象,建立的多重PCR检测技术完全可满足目前检测和监管的需求。主要研究内容和结果如下:(1)转基因水稻转化体特异性多重定性PCR检测技术:针对转基因水稻TT51-1、KMD1、KF6的旁侧序列设计转化体特异性引物,通过引物筛选,多重PCR反应体系和反应条件优化,建立了转基因水稻多重定性PCR检测方法。通过扩增转基因水稻、转基因大豆、转基因玉米、转基因油菜、转基因棉花等不同作物来测试所选择引物的特异性。结果表明,所建立的转基因水稻多重定性PCR检测方法具有较强的特异性,检出限达0.1%。(2)转基因油菜转化体特异性多重定性PCR检测技术:针对转基因油菜RF1、MS8、Topas19/2、Oxy235、T45、GT73、RF2、RF3 品系的旁侧序列设计转化体特异性引物,通过引物筛选,多重PCR反应体系和反应条件优化,建立了转基因油菜多重定性PCR检测方法。经对转基因油菜、转基因大豆、转基因玉米、转基因水稻、转基因棉花等不同作物进行PCR扩增来测试所选择的引物特异性。结果表明,建立的转基因油菜多重定性PCR检测方法具有较强的特异性,检出限为0.05%。(3)转基因油菜转化体特异性多重定量PCR检测技术:针对转基因油菜RF1、Topas19/2、Oxy235、RF3品系的旁侧序列设计转化体特异性引物和探针,通过引物和探针筛选,多重实时荧光定量PCR反应体系和反应条件优化,利用相应标准品梯度稀释绘制标准曲线,确定所建立转基因油菜多重实时荧光定量PCR的检出限。随后采用转基因油菜、转基因大豆、转基因玉米、转基因水稻、转基因棉花等不同代表作物进行PCR扩增来测试所选择的引物和探针的特异性。结果表明,所建立的转基因油菜转化体特异性多重定量PCR方法特异性强和灵敏性高,其检出限为0.05%。
[Abstract]:With the increasing number and variety of genetically modified crops and their products, the potential risks caused by them have attracted much attention.GMO biosafety supervision is related to food safety, food safety and ecological safety. Countries and regions represented by the European Union have issued a series of laws and regulations to strengthen supervision.The detection of transgenic components is an important technical support and means for the safety supervision of transgenic organisms.In recent years, traditional PCR detection technology has been unable to meet the needs of simple, fast and efficient detection of genetically modified components.Because of its high specificity, high sensitivity, low cost, high throughput and saving experimental samples and drugs, multiplex PCR(Multiplex has been widely studied and applied in the field of transgenic component detection.In this paper, the transgenic rice TT51-1KMD1 + KF6 and the transgenic rape RF1MS-8 Topas19 / 2 Oxy235T45 T45 / GT73 RF2RF3 were used as the research objects. The established multiplex PCR detection technology can completely meet the needs of current detection and supervision.The multiplex PCR reaction system and reaction conditions were optimized, and a multiplex qualitative PCR detection method for transgenic rice was established.The specificity of the primers was tested by amplification of transgenic rice, transgenic soybean, transgenic corn, transgenic rapeseed, transgenic cotton and other crops.The results showed that the multiplex qualitative PCR detection method for transgenic rice had strong specificity.A multiplex qualitative PCR detection method for transgenic rapeseed was established.The PCR amplification of different crops such as transgenic rapeseed, transgenic soybean, transgenic corn, transgenic rice and transgenic cotton was carried out to test the specificity of the selected primers.The results showed that the multiplex qualitative PCR detection method for transgenic rapeseed had strong specificity.The reaction system and reaction conditions of multiplex real-time fluorescent quantitative PCR were optimized. The detection limit of multiplex real-time quantitative PCR of transgenic rape was determined by using the standard curve drawn by gradient dilution of the corresponding standard.Then the PCR amplification of different representative crops such as transgenic rape, transgenic soybean, transgenic corn, transgenic rice and transgenic cotton was carried out to test the specificity of the primers and probes selected.The results showed that the established transgenic rapeseed transformant specific multiplex quantitative PCR method had strong specificity and high sensitivity, and its detection limit was 0.05%.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4
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本文编号：1722514