适于转基因水稻RJ5检测的质粒DNA标准物质的研制

Development of Plasmid DNA Reference Material Suitable for Detection of Genetically Modified Rice RJ5

标准物质对于实时荧光定量PCR进行转基因作物及其产品的定量分析尤为重要,但我国转基因生物的标准物质研制还处于初级阶段,因此研制新型的标准物质对解决转基因标准物质匮乏有着重要的意义。本研究针对转基因水稻RJ5构建了质粒标准分子pRJ5,并对其进行定量适应性鉴定、均匀性测试、稳定性考察,并利用数字PCR方法测定其量值。结果表明,以质粒分子pRJ5为标准物质构建的标准曲线具有良好的反应效率和线性相关性及良好的重复性和重演性。在定量PCR体系检测极限(LOD)低至5 copies/μL,定量极限(LOQ)约为10 copies/μL。均匀性测试的数据表明,质粒标准分子在管间和管内无明显差异,具有良好的均匀性。稳定性测试的结果表明,该质粒标准分子至少可以稳定地保存半年以上。数字PCR(3D-d PCR)的量值测定准确度良好。实际样品的定量分析偏差范围在1.31%至2.44%,相对标准偏差值在0.98%至10.05%。以上结果说明了质粒标准分子pRJ5适合作为标准物质对转基因水稻RJ5进行定量检测。

Reference materials are very significant in GMO product quantitative analysis by real-time PCR, however, but the development of GM reference material in our country is still in the initial stage. Therefore, development of novel reference materials can solve the deficiency of reference materials. Herein, we constructed a plasmid reference material pRJ5 corresponding to genetically modified rice R J5 events. And we performed applicability analysis, assessment of homogeneity, stability test by Real-time PCR and measured the certified value of pRJ5 by 3D digital PCR. The results indicated the standard curves using plasmid reference materials pRJ5 as calibrator had high PCR efficiency, good linearity, repeatability and reproducibility. Also pRJ5 had good sensitivity in the qualitative PCR analysis, which LOD was 5 copies per microliter and LOQ was low to 10 copies per microliter. From the results of homogeneity test, plasmid reference material pRJ5 was good in homogeneity. The data of stability test showed that pRJ5 could be stored at least half a year stably. And the results of certified values was ideal by 3D digital PCR. The quantification bias of simulated samples varied from 1.31% to 2.44%, and the relative standard deviations were from 0.98% to 10.05%. In conclusion, the developed plasmid reference material pRJ5 is acceptable and suitable for quantification of the GM rice RJ5.