微滴式数字PCR定量检测转基因玉米品系VCO-01981-5

发布时间：2018-03-04 08:33

本文选题：转基因玉米　切入点：品系VCO--　出处：《食品科学》2017年12期 　论文类型：期刊论文

【摘要】：建立基于QX100微滴式数字聚合酶链式反应(polymerase chain reaction,PCR)平台的我国未批准转基因玉米品系VCO-01981-5的二重微滴式数字PCR定量检测方法。该方法选择基因组中单拷贝的玉米内源基因hmg和VCO-01981-5品系边界序列为定量靶序列,分别设计不同的PCR扩增引物和TaqMan探针,并对两种探针用不同的荧光进行标记,然后将上述探针和引物置于同一个PCR反应体系中以同时定量两个靶标序列。特异性实验结果显示该法只有VCO-01981-5品系的两个靶序列才都有扩增信号。灵敏度、线性和准确性实验结果显示在定量结果相对标准偏差不大于25%时,最低可稳定定量5个拷贝的VCO-01981-5品系特异性序列分子和4个拷贝的内源基因hmg分子;而在高达50 ng模板DNA以下范围内,PCR反应模板量与测定样品拷贝数之间呈高度正相关,相关系数达0.99以上;平均误差小于10%。结果表明本研究建立的该玉米品系定量方法特异性强,稳定性好,精确性、准确性以及灵敏度高,定量范围广,可用于进、出口农产品和食品中该转基因玉米品系成分的定量检测。此外,该法还可为其他转基因玉米品系及其他转基因作物品系建立类似定量检测方法提供参考。
[Abstract]:A double microdrop digital PCR quantitative detection method for transgenic maize strain VCO-01981-5 based on QX100 microdrop polymerase chain reactionation platform was established. The method was used to select the single copy maize endogenous gene in the genome. Because the boundary sequences of hmg and VCO-01981-5 strains are quantitative target sequences, Different PCR amplification primers and TaqMan probes were designed, and the two probes were labeled with different fluorescence. Then the probe and primer were placed in the same PCR reaction system to quantify the two target sequences simultaneously. The specificity experiment showed that only the two target sequences of the VCO-01981-5 strain had amplification signal. The results of linear and accuracy experiments showed that when the relative standard deviation of quantitative results was not greater than 25, the lowest stable quantification of 5 copies of VCO-01981-5 strain specific sequence molecules and 4 copies of endogenous gene hmg molecules; However, within the range of up to 50 ng template DNA, there was a highly positive correlation between the amount of template and the copy number of the sample, the correlation coefficient was more than 0.99, and the average error was less than 10%. The results showed that the quantitative method established in this study had strong specificity. Good stability, accuracy, accuracy and sensitivity, a wide range of quantification, can be used to import and export agricultural products and food components of the transgenic corn strain quantitative detection. The method can also be used as a reference for the establishment of similar quantitative detection methods for other transgenic maize lines and other transgenic crop lines.
【作者单位】：安徽农业大学茶与食品科技院茶树生物学与资源利用国家重点实验室;深圳出入境检验检疫局动植物检验检疫技术中心;深圳市检验检疫科学研究院;
【基金】：公益性行业(质检)科研专项(201410014-2) 深圳市基础研究计划项目(JC201105190969A) 国家质检总局科技司项目(2009IK257)
【分类号】：S513

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