摘要QuantStudioTM 3D 数字PCR (QuantStudioTM 3D digital PCR, 3D-dPCR)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-dPCR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qRT-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，QuantStudioTM 3D 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-dPCR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。
Abstract：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, qPCR 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 (dPCR) makes the accurate and absolute quantification of DNA into reality compared with the qRT-PCR. QuantStudioTM 3D digital PCR (3D-dPCR) 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-dPCR has a huge potential applicability in accurate quantification of GMOs. Herein, we developed the simplex and duplex 3D-dPCR assays to quantify the GM maize (Zea mays) MON863 event, and the results was also compared with those from qRT-PCR. The GM content tested by 3D-dPCR was about half of that tested by qRT-PCR. This because the genotype of GM maize grain was heterozygous. When analyzing the qRT-PCR data we regarded the samples 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 depende 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 3D-dPCR assays showed better consistency than qRT-PCR. The maximum difference between simplex and duplex 3D-dPCR was 13.79%, while the maximum difference between simplex and duplex qRT-PCR was 54.75%. It meaned duplex 3D-dPCR assay was more stable than duplex qRT-PCR assay. Concluded from the results, 3D-dPCR was well developed as one accurate quantification method for GMO products, especially when lacked of the information of sample purity and genotype. Digital PCR could eliminate the influence of these factors and obtain the accurate value. Moreover, digital PCR could also be used as a new method to verify the genotype of GMOs, providing reference for other methods. In addition, compared with qRT-PCR, 3D-dPCR was more suitable for quantitative detection of GMOs by duplex assays. The results, based on 3D-dPCR simplex and duplex quantification methods, provide a good prospect in GMO quantification detection.
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