利用QuantStudio^(TM) 3D数字PCR分析转基因玉米MON863含量  

Quantification of Genetically Modified Maize(Zea mays) MON863 by QuantStudio^(TM) 3D Digital PCR

作　　者：胡佳莹[1] 姜羽[1] 杨立桃[1] 

机构地区：[1]上海交通大学生命科学技术学院,上海200240

出　　处：《农业生物技术学报》2016年第8期1216-1224,共9页Journal of Agricultural Biotechnology

基　　金：转基因生物新品种培育重大专项(No.2016ZX08012-003);教育部新世纪优秀人才计划

摘　　要：Quant StudioTM3D数字PCR(Quant StudioTM3D digital PCR,3D-d PCR)是一种基于超高密度亲疏水微孔芯片实现数字PCR分液原理的新型核酸绝对定量平台,在转基因生物定量领域具有极大的应用前景。本研究基于3D-d PCR平台,以转基因玉米(Zea mays)MON863混合样品为例,建立基于单重和双重数字PCR体系的转基因生物(genetically modified organisms,GMOs)含量分析方法。与传统q RT-PCR比较发现,在缺乏样品纯度、纯合度信息的情况下,数字PCR能够较好地排除这些因素的影响,测定准确的量值。研究结果表明,Quant StudioTM3D数字PCR是一种适用于转基因生物含量分析的精确定量方法,还可反映转基因玉米种子的基因型。本研究基于3D-d PCR建立的转基因玉米MON863单重和双重定量方法为转基因检测提供了新的方法和参考。In the development of nearly 20 years, genetically modified organisms（GMOs） planting area has been growing and the number of GMOs has also been increasing. With the global trade liberalization and commercialization of GMOs, the requirements of analytical approaches also enhances unceasingly. Due to the diversity of genetic modifications in GMOs and low level presence of genetically modified products, the analytical approaches with high sensitivity and accuracy are required to meet the needs of GMO detection.Currently, q PCR is widely used in quantification of GMOs. However, this method relies on the standard curve and reference material, which leads it to a relative quantification method. Digital PCR（d PCR） makes the accurate and absolute quantification of DNA into reality compared with the q RT-PCR. Quant StudioTM3 D digital PCR（3D- d PCR） is a novel absolute quantification method for nucleic acids analysis which based on a hydrophilic and hydrophobic chip with ultra- high density to realize partition. 3D- d PCR has a huge potential applicability in accurate quantification of GMOs. Herein, we developed the simplex and duplex 3D- d PCR assays to quantify the GM maize（Zea mays） MON863 event, and the results was also compared with those from q RT-PCR. The GM content tested by 3D-d PCR was about half of that tested by q RT-PCR, which was because that the genotype of GM maize grain was heterozygous. When analyzing the q RT- PCR data we regarded thesamples and reference materials were homozygous since the genotype of the samples and reference materials were unknown. However, digital PCR was an absolute quantification method which did not depend on any other reference standard, and realized absolute quantification through limit dilution, Poisson distribution and endpoint PCR to ensure the accuracy and reliability of the quantitative results. What′s more, simplex and duplex 3DdP CR assays showed better consistency than qR T-PCR. The maximum difference between simplex and duplex3D-dP CR was 13.79%, whil

关 键 词：QuantStudio^TM 3D数字PCR(3D-d PCR) 转基因生物(GMOs) 精确定量 单重PCR 双重PCR 

分 类 号：Q503[生物学—生物化学]