RPA技术与微滴数字PCR在转基因玉米与大豆检测中的应用

发布时间：2018-04-19 17:56

  本文选题：RPA + ddPCR　； 参考：《上海海洋大学》2017年硕士论文

【摘要】：随着转基因技术的不断发展与应用,转基因产品检测技术的研究对全球转基因产品安全管理至关重要。在这些检测技术中,核酸在恒温下的扩增技术得到了较快的发展。与常规PCR相比,核酸等温扩增技术不再需要热循环仪器,可在恒温条件下快速扩增出目的片段,具有快速、简便、灵敏的优点。其中,重组酶聚合酶扩增技术(Recombinase ploymerase amplification,RPA),是核酸在等温条件下进行扩增的一种技术。微滴数字PCR(droplet digital PCR,ddPCR),是一种核酸检测与定量技术,也是最近几年兴起的新技术。本研究的内容和结果如下:1、RPA结合凝胶检测技术特异性检测转基因玉米Bt11:本研究根据转基因玉米Bt11外源基因草丁膦乙酰转移酶基因(phosphinothricin acetyl transferase gene,PAT)及载体骨架连接区序列,设计引物,首次建立了基于RPA与凝胶检测技术相结合的方法,特异性地快速检测转基因玉米Bt11,在37℃条件下20min内即可完成检测。本实验所得该方法的绝对检测限约为100拷贝,相对检测限约为0.1%。2、RPA结合荧光检测技术检测转基因玉米Bt11外源基因nos终止子:此研究选用转基因玉米Bt11为材料,研究RPA荧光技术检测外源基因nos终止子的灵敏度,测得绝对和相对检测限分别约为50拷贝和0.1%。与之对比实验,本实验采用荧光定量PCR染料法检测转基因玉米Bt11外源基因nos终止子,实验测得绝对检测限达10拷贝甚至以下,相对检测限为0.1%。荧光定量PCR可对转基因玉米含量进行定量测定,但荧光定量PCR相对检测时间较长,需要两小时以上,并且定量时需要做标准曲线,过程复杂,而应用RPA荧光技术检测时间明显缩短,半小时就可以完成检测。两种技术各有优势,可根据实际需要选取。3、ddPCR测定转基因大豆GTS-40-3-2转基因成分含量:在本研究中,使用了ddPCR和荧光定量PCR对10%的转基因大豆GTS-40-3-2标准品进行了转基因成分含量的测定,两种方法测得转基因含量与实际相符,都约为10%。相比ddPCR方法,荧光定量PCR需要更多的模板量和引物,加样的数量也比较多。采用QX200TM Droplet Digital TM PCR系统,将两套引物和探针加入到同一PCR管中,减少了加样数量,也节省了模板量。在数据分析方面,荧光定量PCR方法需要根据荧光数值构建标准曲线,并通过计算获得基因的拷贝数和转基因成分的含量,而ddPCR不依赖标准曲线,能够通过不同荧光的微滴数目直接获得靶基因的拷贝数,并精确计算出转基因成分所占的百分数。本实验所得ddPCR方法的最低检出限约为2个拷贝。
[Abstract]:With the development and application of transgenic technology, the research of transgenic product detection technology is very important to the global safety management of transgenic products.Among these detection techniques, nucleic acid amplification at constant temperature has been developed rapidly.Compared with conventional PCR, the nucleic acid isothermal amplification technique no longer requires a thermal cycle instrument, and can rapidly amplify the target fragment under the condition of constant temperature, which has the advantages of rapidity, simplicity and sensitivity.Among them, Recombinase ploymerase amplification is a technique for nucleic acid amplification under isothermal conditions.Microdrop digital PCR(droplet digital rcad PCR is a nucleic acid detection and quantification technique, and it is also a new technology developed in recent years.The contents and results of this study were as follows: Bt11 of transgenic maize was specifically detected by the combination of 1: 1 RPA and gel detection. Primers were designed based on phosphinothricin acetyl transferase gene (PATT) and the sequence of vector skeleton junction region (PATs) of transgenic maize Bt11.For the first time, a method based on RPA and gel detection was established to detect transgenic maize BT 11 specifically and quickly, which could be detected in 20min at 37 鈩，

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