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首页>《中国测试》期刊>本期导读>转基因玉米MON89034、MON810、MIR162双重数字PCR定量方法的建立

转基因玉米MON89034、MON810、MIR162双重数字PCR定量方法的建立

75    2019-06-26

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作者:梁文, 杨镇州, 李妍, 罗超, 闻艳丽, 刘刚

作者单位:上海市计量测试技术研究院, 上海 201203


关键词:生物技术;数字PCR;双重数字PCR;转基因玉米


摘要:

针对进出口贸易中涉及较多的转基因玉米MON89034、MON810、MIR162品系,研究一套双重数字PCR(dPCR)定量检测方法,包括引物探针的序列设计和浓度,DNA模板浓度,PCR反应过程的时间、温度等。该方法的定量限为0.1%,转基因定量检测范围覆盖0.1%~100%,线性系数为0.999,精密度优于10%。该检测方法中,每个微反应体系都含有两套引物探针,分别用FAM和VIC荧光通道进行检测,能实现内外源基因的同时检测,避免同一样品因取样不一致造成的定量差异。该方法可以同时应用到市面上最常用微滴dPCR平台和3D芯片dPCR平台,且两种方法定量结果一致性好。


A quantitative method for genetically modified maize event MON89034, MON810, MIR162 using duplex digital PCR
LIANG Wen, YANG Zhenzhou, LI Yan, LUO Chao, WEN Yanli, LIU Gang
Shanghai Institute of Measurement and Testing Technology, Shanghai 201203, China
Abstract: This paper reports a quantification method for genetically modified maize, MON89034, MON810, MIR162 based on duplex digital PCR (dPCR). Several important analysis conditions of dPCR were formed including sequence designing of primers and probes, the template concentration range, the duration time and temperature of PCR reaction. The limit of quantification of the method was 0.1%, and the quantitative detection range covered 0.1%-100% with a linear coefficient 0.999, and the precision was better than 10%. In this method, each micro-reaction system contains two sets of primer probes, which are respectively detected by FAM and VIC fluorescence channels, and the internal and external sequences can be detected simultaneously, thereby avoiding the quantitative error caused by the sampling inconsistency. The method was demonstrated on two main dPCR platform:microdrop dPCR and chip-based dPCR, and both of the quantitative results were consistent.
Keywords: biotechnology;digital PCR;duplex digital PCR;genetically modified maize
2019, 45(6):70-76  收稿日期: 2019-01-02;收到修改稿日期: 2019-03-18
基金项目: 国家科技重大专项项目(2018ZX0801112B);国家自然科学面上基金项目(21775104);上海市质量技术监督局科研项目(2017-03)
作者简介: 梁文(1986-),女,四川德阳市人,工程师,硕士,主要从事转基因检测方法研究、核酸定量方法研究
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