转基因大豆MON89788芯片式数字PCR定量方法的建立

针对抗虫耐除草剂大豆转基因品系MON89788,从转基因植物基因组DNA的提取、核酸模板的质量和浓度控制、引物探针的筛选、PCR反应过程的建立等方面建立了一套完整的转基因大豆芯片式dPCR定量检测方法。本实验也对该方法的重复性和定量检测限进行考察。10组5%转基因品系大豆MON89788样品定量重复性RSD在1.17%-9.97%之间,均满足国际上转基因定量结果RSD小于25%的要求。用该方法对转基因含量为5%、1%、0.1%的大豆MON89788进行定量检测,其定量结果为5.20%、0.94%和0.11%,RSD分别为6.2%、3.6%和15.2%。该检测方法的定量限达到0.1%,能满足欧盟对转基因定量标识0.9%的要求。将本实验建立的方法用于转基因大豆的定量检测,能为规范我国转基因监管工作的实施提供强有力的技术支撑。

A Quantitative Method for Genetically Modified Soybean LineMON89788 Using Microchip Digital PCR

A microchip digital PCR(dPCR)quantitative method for insect-resistant and herbicide-resistant genetically modified soybean lineMON89788was developed and several critical experimental conditions were studied,including genomic DNA extraction,quality and concentration control of nucleic acid template,screening of the primer probes,PCR program,etc.The reproducibility and quantitative detection limit of the method were also investigated.The quantitative repeatability RSD ranged from 1.17% to 9.97% when a 5% GM soybean(MON89788)sample was analyzed,andall of them met the international requirement that the RSD of transgenic quantitative results should be < 25%.The quantitative results of MON89788samples containing 5%,1% and 0.1% GM were 5.20%,0.94% and 0.11% and the RSD was 6.2%,3.6% and 15.2%,respectively.The limit of quantificationwas 0.1%,which fully satisfiedthe EU’s GM quantitative labeling limit(0.9%).Applying the method established in this experiment in the quantitative detection of transgenic soybeans may provide strong technical support for standardizing the implementation of transgenic supervision in China.