转基因康乃馨Moonlite品系检测用质粒标准样品研制

Development of plasmid standard material for detection of genetically modified carnation line Moonlite

截至2020年底,国际上已有19种转基因康乃馨品系进入商业化种植。为了实现转基因康乃馨的有效检测监管,研制检测标准样品十分必要。目前,国际上还没有该类标准品的相关报道。为此,本文研制了商业化时间最长、种植量较大的转基因康乃馨Moonlite品系质粒标准样品,使其与转基因康乃馨检测技术标准的应用相配套,为口岸转基因产品的检测监管提供必要的技术支撑。首先,将康乃馨内源ANS基因1279 bp片段和转基因康乃馨Moonlite品系406 bp 5′端特异性序列分别插入到pcDNA3.0载体的酶切位点和距离1 kb以上的多克隆位点上,合成质粒DNA标准样品。后将质粒DNA标准样品进行稀释、分装,对其均匀性、最小取样量和短期、长期稳定性等进行测定,采用数字PCR方法对质粒DNA标准样品进行定值,并进行不确定度评估。质粒DNA标准样品pLite经全基因组测序,与公开发布的序列完全一致。均匀性实验结果表明,分别以ANS基因和Moonlite品系特异性序列为目标进行测定,F值分别为1.61和1.14,均小于95%置信区间的F值,质粒DNA标准样品足够均匀。质粒DNA标准样品的最小取样量为2μL。稳定性实验结果表明,质粒DNA标准样品pLite在45℃高温条件下,可稳定保存14 d;在26℃及以下温度条件下,可稳定保存1个月,在4℃及以下温度可稳定保存6个月,说明质粒DNA标准样品适宜运输。质粒DNA标准样品在反复冻融25次后,拷贝数浓度无显著变化。定值结果表明,质粒DNA标准样品pLite中康乃馨内源ANS基因拷贝数的参考值为(6.13±0.22)×10^(6)μL^(-1),Moonlite品系特异性序列拷贝数的参考值为(6.10±0.24)×10^(6)μL^(-1)。研制的质粒DNA标准样品pLite具有良好的均匀性和稳定性,获得国家标准样品证书后,可用于转基因康乃馨Moonlite品系的检测。

By the end of 2020 year,nineteen genetically modified(GM)carnation lines had been commercialized in the world.In order to detect and supervise GM carnations effectively,it is necessary to develop standard materials.Until now,there are no standard materials for GM carnation detection that have been reported around the world.Therefore,this study developed a plasmid standard molecule pLite for GM carnation line Moonlite,which has been commercialized and planted for a long time,so that it could be suitable for the application of the standard for GM carnation detection,and could support the inspection and supervision of GM products imported and exported.To develop a plasmid DNA molecule for Moonlite,two fragments of carnation endogenous ANS gene with 1279 bp in length and the 5′-end event-specific sequence of GM carnation Moonlite with 406 bp in length were inserted into plasmid pcDNA3.0.The distance between the two inserted sites was more than 1 kb.Then,the plasmid DNA was diluted and packaged.The homogeneity,minimum sampling amount,short-term and long-term stability and reference values of the plasmid were measured,meanwhile,the uncertainty were evaluated.The genome sequencing results showed that the sequence of pLite was consistent with the published sequence.Results of the homogeneity test showed that F values of ANS gene and Moonlite event-specific sequence were 1.61 and 1.14,respectively,which were both less than the F value under 95%confidence interval,indicating that the plasmid DNA molecule was homogeneous.The minimum sampling amount of pLite was 2μL.Results of stability experiments showed that pLite stayed stable at 45℃for 14 days.At room temperature of 26℃and below,the plasmid DNA molecule could be stably stored for one month,and could be stably stored for 6 months at 4℃and below,indicating that pLite was suitable for transportation.After repeated freeze-thaw 25 times,the copy number of pLite had no significant change,which could meet the requirements of daily detection.The calculation results indicated that the reference values of ANS gene and Moonlite specific sequence were(6.13±0.22)×10^(6)μL^(-1) and(6.10±0.24)×10^(6)μL^(-1) in pLite,respectively.The developed plasmid DNA molecule pLite had good homogeneity and stability,at the same time it was suitable for the detection of GM carnation event Moonlite.