目的 建立檢測地特胰島素原料中宿主DNA殘留量的熒光定量核酸擴增（Real-time PCR）方法并進行驗證，用于該產(chǎn)品的質(zhì)量控制。方法 選擇釀酒酵母5sRNA作為靶基因設(shè)計引物，提取地特胰島素原料中的殘留宿主DNA，采用Real-time PCR SYBRGreen染料法對標準DNA和樣品進行測定，繪制標準曲線并分析樣品中的DNA殘留量。對建立的方法進行方法學(xué)驗證，并測定3批地特胰島素原料中的殘留宿主DNA。結(jié)果 釀酒酵母基因組DNA質(zhì)量濃度在0.18~180 000 ng/mL線性良好（r²=0.998 5）；回收率均在80.0%~106.3%，檢測3批地特胰島素原料的宿主DNA殘留量均低于進口藥品注冊標準限度。結(jié)論 該方法可用于釀酒酵母生產(chǎn)的地特胰島素原料中殘留宿主DNA的定量測定。

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