目的 建立检测地特胰岛素原料中宿主DNA残留量的荧光定量核酸扩增（Real-time PCR）方法并进行验证，用于该产品的质量控制。方法 选择酿酒酵母5sRNA作为靶基因设计引物，提取地特胰岛素原料中的残留宿主DNA，采用Real-time PCR SYBRGreen染料法对标准DNA和样品进行测定，绘制标准曲线并分析样品中的DNA残留量。对建立的方法进行方法学验证，并测定3批地特胰岛素原料中的残留宿主DNA。结果 酿酒酵母基因组DNA质量浓度在0.18~180 000 ng/mL线性良好（r²=0.998 5）；回收率均在80.0%~106.3%，检测3批地特胰岛素原料的宿主DNA残留量均低于进口药品注册标准限度。结论 该方法可用于酿酒酵母生产的地特胰岛素原料中残留宿主DNA的定量测定。

Objective To establish a Real-time PCR method for quantitative detection of Saccharomyces cerevisiae nucleotide residues in insulin detemir substances, to control the quality of insulin detemir substances. Methods The 5S ribosomal RNA gene of Saccharomyces cerevisiae was used as target gene to design primer, and the residual host cell DNA was extracted and determined by SYBRGreen based q-PCR. The residual host cell DNA was analyzed according to the standard curve. The developed method was verified and used for determination of 3 batches of insulin detemir substances. Results The calibration curve was linear over the range of 0.18-180 000 ng/mL, the correlation coefficient r² was 0.998 5, and the recovery rates of spiked samples with different DNA quantity were between 80.0%-106.3%. The residual host cell DNA determined by this method was not more than the limit, which was adopted by imported drug registration standards. Conclusion The method can be used for the quantitative determination of Saccharomyces cerevisiae nucleotide residues in insulin detemir substances.

R927.2