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.
HU Jia-Ying,JIANG Hu,YANG Li-Tao. Quantification of Genetically Modified Maize (Zea mays) MON863 by QuantStudioTM 3D Digital PCR[J]. , 2016, 24(8): 1216-1224.
[1]Heid C A, Stevens J, Livak K J, et al.Real time quantitative PCR[J].Genome research, 1996, 6(10):986-994 [2]Cankar K, ?tebih D, Dreo T, et al.Critical points of DNA quantification by real-time PCR–effects of DNA extraction method and sample matrix on quantification of genetically modified organisms[J].BMC biotechnology, 2006, 6(1):37-52 [3]Morisset D, Stebih D, Milavec M, et al.Quantitative analysis of food and feed samples with droplet digital PCR[J].PLoS One, 2013, 8(5):e62583- [4]Sykes P J, Neoh S H, Brisco M J, et al.Quantitation of targets for PCR by use of limiting dilution[J].Biotechniques, 1992, 13(3):444-449 [5]Burns M J, Burrell A M, Foy C A.The applicability of digital PCR for the assessment of detection limits in GMO analysis[J].European Food Research and Technology, 2010, 231(3):353-362 [6]Song L.Application of digital PCR in analyzing transgenic soybeans[C]. Plant and Animal Genome XXIII Conference. Plant and Animal Genome. 2015. [7]Mead E, Wang Y, Patricia H, et al.Precise and Accurate Determination of microRNA Precursors by Digital PCR[J].Journal of biomolecular techniques, 2014, 25(Suppl):S27- [8]袁亚男, 刘文忠.实时荧光定量 技术的类型, 特点与应用[J].中国畜牧兽医, 2008, 35(3):27-30 [9]杨立桃, 赵志辉, 丁嘉羽, 等.利用实时荧光定量方法分析转基因水稻外源基因拷贝数[J].中国食品卫生杂志, 2005, 17(2):142-144 [10]Pinheiro L B, Coleman V A, Hindson C M, et al.Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification[J].Analytical chemistry, 2011, 84(2):1003-1011 [11]林彩琴, 姚波, 靳玉慎, 等.数字 技术进展[J].化学进展, 2012, 24(012):2415-2423 [12]姜羽, 胡佳莹, 杨立桃.利用微滴数字 分析转基因生物外源基因拷贝数[J].农业生物技术学报, 2014, 22(10):1298-305 [13]European network of GMO laboratories.Definition of minimum performance requirements for analytical methods of GMO testing. 2008, http://gmo-crl.jrc.it. [14]Trifa Y, Zhang D.DNA content in embryo and endosperm of maize kernel (Zea mays L): impact on GMO quantification[J].Journal of agricultural and food chemistry, 2004, 52(5):1044-1048
