中国仓鼠卵巢细胞DNA国家标准品的研制

Preparation of National Standard Substance of CHO Cell DNA

目的建立中国仓鼠卵巢细胞DNA残留量定量测定用国家标准品。方法使用QIAGEN Genomic-tip 500/G以及基因组DNA纯化试剂制备了中国仓鼠卵巢细胞基因组DNA作为标准品原料,通过紫外分光光度法和电泳进行纯度和含量测定后分装,采用紫外分光光度法对我国第一批中国仓鼠卵巢细胞DNA国家标准品进行协作标定,并评价其稳定性和适用性。结果中国仓鼠卵巢细胞DNA国家标准品经检测,A260/A280均在1.7～1.9之间,电泳图谱只有单一条带,符合规定。该标准品经6家实验室协作标定共90次,几何平均含量为93.63μg.mL-1,平均含量的95%可信区间为92.86～94.40μg.mL-1,单次测定的95%参考值范围为86.51～100.89μg.mL-1,平均可信限率为0.81%。加速热稳定实验表明,该标准品在-20、4、25、37℃条件下放置4个月,DNA含量无明显改变,但37℃条件下A260/A280有下降趋势;-20、4、25℃条件下电泳结果为单一条带,37℃条件下DNA有降解条带。-20℃贮存1年的长期稳定性结果显示,标准品DNA浓度及A260/A280无显著性差异,电泳条带单一,因此-20℃条件下长期保存标准品稳定性较好。在标准品适用性研究中,利用该标准品进行荧光染色法测定,标准曲线r值为0.999 0以上,在0.781～100 ng.mL-1之间线性良好,各稀释度RSD均小于10%;利用该标准品进行定量PCR法测定,标准曲线r值为0.990 0以上,在10-2～103pg之间线性良好,熔解曲线可见单一峰出现。结论该批中国仓鼠卵巢细胞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染色法和定量PCR检测中国仓鼠卵巢细胞DNA残留量,含量定为93.63μg.mL-1,批号为270026-201101。

OBJECTIVE To prepare the national standard substance for quantitative determination of residual DNA in CHO cells. METHODS CHO cell DNA was prepared using QIAGEN Genomic-tip 500/G and genomic DNA purification reagents and an- alyzed for purity and concentration by UV speetrophotometry and agarose gel electrophoresis. Then the standard substance of CHO DNA was calibrated collaboratively, and evaluated for stability and applicability. RESULTS The prepared national standard substance of CHO DNA was qualified as indicated by A26o/A28o between 1.7 and 1.9 and a single specific band in agarose gel electrophoresis. The standard substance was calibrated for 90 times by six laboratories, and the results showed a geometric mean concentration of 93.63 μg· mL-1 （95% confidence interval 92. 86-94.40 μg · mL-1 ）. The 95% confidence interval of the geometric mean concentration in a single determination was 86. 51 - 100. 89 μg · mL-1. The mean confidence limit rate was 0. 81%. The DNA concentration was stable after storage at - 20, 4, 25 and 37 ℃ for 4 mouths, but A260/A280 was decreased when stored at 37 ℃ for 4 months. The elec-trophoresis results showed a single band after storage at - 20, 4 and 25 ℃ for 4 months, but showed degradation after storage at 37 ℃ for 4 months. The long term stability test revealed that the DNA concentration and purity were stable after storage at -20 ℃ for 12 months. In applicability studies using the CHO DNA standard substance, the fluorescence method showed good finearity （r 〉 0. 990 0） in the concentration range of 0. 781 - 100 ng · mL-1, with RSD of less than 10%. The real-time PCR had high sensitivity up to 10-2 pg of DNA with good linearity （r 〉0. 990 0） in the content range of 10-2 - 10^3pg, and the melting curve showed a single peak. CONCLUSION The prepared standard substance with batch number of 270026-201101 and DNA concentration of 93.63 μg · mL-1 is qualified in overall tests and may be used as national standard substance for residual DNA assay by fluorescence and real-time PCR methods.