日常豆制品中抗草甘膦大豆转基因成分的Realtime-PCR检测方法的建立和应用

发布时间：2018-07-03 02:48

本文选题：抗草甘膦转基因大豆 + Realtime-PCR　；参考：《北京工业大学》2016年硕士论文

【摘要】：本论文以大豆及其加工产品为对象,建立了用于快速检测豆制品中转基因大豆成份——抗草甘膦基因EPSPS及其表达元件的Realtime-PCR检测法,并对市场上常见豆制品进行检测分析。该方法的建立,可为食品监管机构有效的管理转基因产品提供理论和实验依据和参考。聚乙烯吡咯烷酮(PVP)浓度对大豆基因组DNA提取的影响:对来自三个地区的大豆样本进行了分析,并对其中看家基因Lectin基因进行PCR反应和凝胶电泳分析,结果显示,0.1%以上PVP能有效的提高大豆基因组DNA提取质量,凝胶电泳分析结果中能够明显看到目标基因所在位置,条带清晰。酱油中抗草甘膦转基因大豆成分检测方法的建立:建立了利用RT-PCR反应(SybGreen法)对酱油中提取出的基因组DNA进行检测的方法,通过对提取出的基因组DNA进行RT-PCR检测分析显示,在以转基因豆粕为原料的酱油所提取出的基因组DNA中能够检测到CaMv35s启动子和Nos终止子调控元件,而外源基因EPSPS基因降解较严重,熔解曲线分析表明,RTPCR对转基因的检测获得了很好的特异性。RT定量PCR相对于传统PCR,具有快速、高通量、高选择性、高灵敏度的优势,在转基因食品的检测领域具有很大的潜力。市场上常见豆制品中抗草甘膦转基因大豆成分的检测:以自农贸市场的散装豆制品和来自超市中的袋装豆制品共28种为检测对象,利用RTPCR反应对其中抗草甘膦转基因大豆成分进行了检测。结果发现,11种袋装豆干类产品中有2种检测到抗草甘膦转基因大豆CaMv35s启动子、Nos终止子和EPSPS基因,1种检测到抗草甘膦转基因大豆CaMv35s启动子和Nos终止子。17种散装豆制品中只有1种检测到CaMv35s启动子、Nos终止子和EPSPS基因。28种样品中,在标签中明确标注原料为非转基因大豆的产品都未检测出抗草甘膦转基因大豆成分,未标注是否含转基因大豆原料的产品中部分检测出4种含抗草甘膦转基因大豆成分。
[Abstract]:In this paper, we set up a Realtime-PCR detection method for fast detection of genetically modified soybean components, glyphosate resistance gene EPSPS and its expression components in soybean products, and analyzed the common soybean products in the market. The establishment of this method can effectively manage GM production for food regulators. The effects of the concentration of polyvinylpyrrolidone (PVP) on soybean genomic DNA extraction were provided. The soybean samples from three regions were analyzed, and the PCR reaction and gel electrophoresis of the Lectin gene were analyzed. The results showed that more than 0.1% PVP could effectively improve the DNA extraction of soybean genome. The location of the target gene can be clearly seen in the analysis results of the gel electrophoresis. The method for detecting the composition of glyphosate resistant transgenic soybean in soy sauce is established: the method of detecting genomic DNA extracted from soy sauce by RT-PCR reaction (SybGreen) is established, and RT-PC is carried out by the extraction of genomic DNA. R detection analysis showed that CaMv35s promoter and Nos terminator were detected in the genomic DNA extracted from soy sauce with genetically modified soybean meal, and the exogenous gene EPSPS gene was degraded seriously. The analysis of the fusion curve showed that RTPCR obtained a good specific.RT quantitative PCR relative to the traditional PCR for the detection of transgene. With the advantages of rapid, high throughput, high selectivity and high sensitivity, it has great potential in the field of detection of genetically modified foods. The detection of glyphosate resistant transgenic soybean components in common soybean products on the market: 28 kinds of samples from the bulk soybean products and the bags from the supermarkets from the farmers' market and the RTPCR reaction The results showed that 2 kinds of glyphosate resistant transgenic soybean CaMv35s promoter, Nos terminator and EPSPS gene were detected in 11 bags of soybean stem, and only 1 detected C in 1 kinds of glyphosate transgenic soybean CaMv35s promoter and Nos terminal.17 bulk soybean products. In the aMv35s promoter, the Nos terminator and the EPSPS gene.28 samples, there was no detection of glyphosate resistant genetically modified soybean ingredients in the label which was clearly labeled as non GM soybean, and 4 kinds of transgenic soybean components containing glyphosate were detected.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TS214

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