NSO细胞DNA首批国家标准品的建立

Establishment of the First National Standard of NSO Cell DNA

目的建立小鼠骨髓瘤NSO细胞DNA含量测定国家标准品。方法使用QIAGEN基因组DNA纯化试剂以及Genomictip 500/G制备了NSO细胞DNA作为标准物质原料。通过紫外分光光度法和琼脂糖凝胶电泳进行纯度和含量测定,每支100μL分装于螺口冻存管。组织5个独立实验室应用紫外分光光度法对我国第一批NSO DNA国家标准品进行协作标定,并评价DNA标准物质的稳定性与适用性。结果所制备的NSO细胞DNA标准品经检测,A260/A280均在1.7~1.9之间,电泳图谱条带单一,纯度符合要求。该DNA标准品经5家实验室协作标定共90次,几何平均含量为87.5μg·mL^-1,平均含量的95%可信区间为86.6~88.5μg·mL^-1。短期稳定性实验表明,该标准品在4℃条件下放置4个月,DNA标准品浓度测定值及/1260/无显著性差异;-20、-70℃贮存1年的长期稳定性结果显示,标准品DNA浓度测定值及A260/A280无显著性差异,电泳条带单一,因此该标准物质应在-20℃条件以下长期保存。在标准品适用性研究中,利用该标准品进行定量PCR法测定,在3 fg·μL^-1~300 pg·μL^-1之间线性良好,标准曲线r^2值>0.999。结论该批NSO 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定量PCR法检测NS0细胞DNA残留量,DNA标准品含量赋值为87.5μg·mL^-1,批号为330002-201701。

OBJECTIVE To prepare the national standard substance for quantitative determination of residual DNA in mouse myeloma(NSO)cells.METHODS NSO cell DNA was prepared using genomic DNA purification reagents QIAGEN and Genomic-tip 500/G,analyzed for purity and concentration by UV spectrophotometry and agarose gel electrophoresis,and split in 100 microliters and frozen in screw cap tubes.Then five independent laboratories were organized to calibrate the first batch of NSO DNA national standard using UV spectrophotometry,and evaluated the stability and applicability.RESULTS The prepared national standard substance of NSO DNA was qualified as indicated by A260/A280 between 1.7 and 1.9 and a single specific band in agarose gel electrophoresis.The standard substance was calibrated for 90 times by the five laboratories,and the results showed a geometric mean concentration of 87.5μg·mL^-1,the 95%confidence interval of the geometric mean concentration in a single determination was 86.6-88.5μg·mL^-1.Short-term stability experiments showed that there was no significant change in the standard DNA concentration and A260/A280 ratio after being stored at 4℃ for 4 m.The results of storage stability test at-20 and-70 G for 1 year revealed that there was no significant change in the standard DNA concentration and A260/A280 ratio,and the electrophoresis strip was single,so the standard substance was stable at-20℃.In applicability studies using the NSO DNA standard substance,the real-time PCR had high sensitivity up to 0.003 pg of DNA with good linearity(r^2>0.999)in the content range of 3 fg·μL^-1-300 pg·μL^-1.CONCLUSION The prepared standard substance with batch number of 330002-201701 and DNA concentration of 87.52μg·mL^-1 is qualified in all tests and may be used as national standard substance for residual DNA assay by real-time PCR method.