首批HEK293细胞DNA含量测定国家标准品的制备  

Preparation of the first national reference standard for determination of HEK293 cell DNA content

作　　者：王光裕[1] 杨靖清[1] 魏长龙 周泽鑫 杨志行 黄钰 周勇[1] WANG Guangyu;YANG Jingqing;WEI Changlong;ZHOU Zexin;YANG Zhixing;HUANG Yu;ZHOU Yong(National Institutes for Food and Drug Control,National Key Laboratory of Pharmaceutical Regulatory Science,Beijing 100050,China;不详)

机构地区：[1]中国食品药品检定研究院药品监管科学全国重点实验室,北京100050 [2]广州派真生物技术有限公司,广东广州510670 [3]湖州申科生物技术有限公司,浙江湖州313000 [4]武昌首义学院,湖北武汉430064

出　　处：《中国生物制品学杂志》2024年第3期316-321,共6页Chinese Journal of Biologicals

基　　金：北京市科技计划(Z2211000079220155);2022年度国家药品标准制修订研究课题(2022S07)。

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160μL/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103.8~105.8 ng/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0.3~3000 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R^(2)分别为0.9992和0.9995。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。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 approximately 100 ng/μL,the DNA was aliquoted at 160μL/tube.Five different laboratories were organized for collaborative calibration by using ultraviolet spectrophotometry,and the stability and applicability were evaluated.Results The HEK293 cell DNA national reference standard exhibited A_(260)/A_(280)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 interval for the mean was 103.8—105.8 ng/μL,and the 95%reference range for single measurements was 96.0—113.6 ng/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—3000 pg/reaction,with amplification efficiencies of 101%and 95%,and R^(2) values of 0.9992 and 0.9995 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.

关 键 词：HEK293细胞 宿主残余DNA 国家标准品 荧光定量PCR 

分 类 号：R917[医药卫生—药物分析学]