摘要
目的 制备HEK293细胞DNA含量测定用国家标准品,以期为行业内HEK293细胞残余DNA的测定提供支持.方法 使用Genomic-tip 500/G以及基因组DNA纯化试剂制备HEK293细胞DNA,以其为标准物质原料,采用紫外分光光度法和琼脂糖凝胶电泳法进行纯度和含量筛选,随后稀释至浓度约为100 ng/μL后,按160 μL/支进行分装.组织5家不同实验室,采用紫外分光光度法进行协作标定,并评价其稳定性和适用性.结果 制备的HEK293细胞DNA国家标准品经检测,A260/A280均在1.8～2.0之间,电泳图谱为单一条带,符合规定.该标准品经5家实验室协作标定,共得到78个有效数据,平均含量为104.8 ng/μL,相对标准偏差(relative standard deviation,RSD)为4.2％,均数的95％可信区间为103.8～105.8ng/μL,单次测定的95％参考值范围为96.0～113.6 ng/μL,平均可信限率为1.0％,推荐保存条件为-80 ℃.采用2个不同型号的荧光定量PCR仪进行适用性研究,该标准品在0.3～3 000 pg/反应范围内适用性良好,扩增效率分别为101％和95％,标准曲线R2分别为0.999 2和0.999 5.结论 该批HEK293细胞DNA国家标准品各项指标均符合要求,可作为国家标准品用于荧光定量PCR检测,含量定为 104.8 ng/μL,批号为270039-202301.
Abstract
Objective To prepare a national reference standard for the quantification of HEK293 cell DNA content,so as to provide a support for the determination of residual DNA in HEK293 cells in the industry.Methods HEK293 cell DNA prepared using Genomic-tip 500/G and genomic DNA purification reagents was used as source materials,and the purity and content were assessed using ultraviolet spectrophotometry and agarose gel electrophoresis.After dilution to app-roximately 100 ng/μL,the DNA was aliquoted at 160 μL/tube.Five different laboratories were organized for collabora-tive calibration by using ultraviolet spectrophotometry,and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A260/A280 ratios between 1.8 and 2.0 and displayed a single band on electrophoresis,meeting the specified criteria.Collaborative calibration across five laboratories yielded 78 valid data points with an average content of 104.8 ng/μL,a relative standard deviation(RSD)of 4.2％.The 95％confidence inte-rval for the mean was 103.8-105.8ng/μL,and the 95％reference range for single measurements was 96.0-113.6ng/μL.The average confidence limit rate was 1.0％,and the recommended storage condition was-80 ℃.Applicability studies were conducted using two different models of fluorescence quantitative PCR instruments.The reference standard exhibited good applicability within the range of 0.3-3 000 pg/reaction,with amplification efficiencies of 101％and 95％,andR2 values of 0.999 2 and 0.999 5 for the standard curves,respectively.Conclusion This batch of HEK293 cell DNA national reference standard meets all required specifications and can be utilized as a national reference standard for fluorescence quantitative PCR detection,with a certified content of 104.8 ng/μL,assigned batch number 270039-202301.
基金项目
北京市科技计划(Z2211000079220155)
国家药品标准制修订研究课题(2022)(2022S07)
NETL
NSTL
万方数据