蛋白药物生产中支原体检测方法的建立及应用  

Development of a Detctection Method for Mycoplasma in Manufacture of Recombinant Therapeutic Proteins

作　　者：刘素霞[1] 刘晓志[1] 杨立霞[1] 赵伟[1] 常亮[1] 周兴军[1] 高健[1] 

机构地区：[1]华北制药集团新药研究开发有限责任公司,抗体药物研制国家重点实验室,石家庄050015

出　　处：《生物技术进展》2013年第4期293-296,F0003,共5页Current Biotechnology

基　　金：国家863计划项目(2012AA02A306);国家973计划项目(2012CB724502)资助

摘　　要：为检测中国仓鼠卵巢细胞(CHO)细胞培养过程中的支原体污染,以CHO细胞的管家基因甘油醛-3-磷酸脱氢酶作为内参基因,将内参引物和支原体引物同时放置在一个PCR体系中,确立了一种快速、准确、稳定的PCR检测方法。通过优化两种引物的浓度配比,确定内参基因引物浓度为5μmol/L,支原体引物浓度为10μmol/L。与DNA荧光染色法相比,PCR法检测结果一致,减少了假阴性结果,缩短了检测时间,可以满足生产过程中的支原体污染的监测。For the detection of mycoplasma contamination in Chinese hamster ovary cells （CHO） cell culture, a rapid, accurate, and stable PCR detection method was established. A housekeeping gene encoded glyeeraldehyde 3-phosphate dehydrogenase was used as a reference gene, and the internal reference primers and primers for mycoplasma detection were placed in a PCR system simultaneously. The concentration ratio of two kinds of primers was optimized as 5 ~Lmol/L reference gene primers and 10 μmol/L mycoplasma detection primers. PCR method had consistent results with DNA staining method, with less false negative result and short detction time, showing that it could meet the demand for myeoplasma contamination detection in the production process

关 键 词：中国仓鼠卵巢细胞 支原体 PCR反应 

分 类 号：S859[农业科学—临床兽医学]