实时荧光定量PCR法检测外源性DNA在重组Ⅲ型人源化胶原蛋白冻干纤维中的残留  

Detection of exogenous DNA residues in recombinant type Ⅲ humanized collagen lyophilized fibers by real-time fluorescence quantitative PCR

作　　者：梁凯歌 孙丹丹 LIANG Kai-ge;SUN Dan-dan(Shanxi Key Laboratory of Functional Protein,Shanxi Jinbo Biomedical Co.,Ltd.,Taiyuan 030000,China)

机构地区：[1]山西锦波生物医药股份有限公司功能蛋白山西省重点实验室,太原030000

出　　处：《中国医药生物技术》2023年第1期30-34,共5页Chinese Medicinal Biotechnology

摘　　要：目的对实时荧光定量PCR(qPCR)法检测重组Ⅲ型人源化胶原蛋白冻干纤维中的外源性DNA残留量进行探究和分析,确定该方法用于DNA质量控制的可行性。方法采用宿主细胞残留DNA样本前处理试剂盒(磁珠法)提取DNA,利用E.coli残留DNA检测试剂盒(PCR-荧光探针法)进行扩增反应,绘制标准曲线,建立重组Ⅲ型人源化胶原蛋白冻干纤维中外源性DNA残留量的qPCR检测方法。结果对8批重组Ⅲ型人源化胶原蛋白冻干纤维进行测定,外源性DNA残留量在0.03~300 pg/μl内,重复性良好,均远低于每剂10 ng,线性良好(R^(2)>0.99),回收率均在50%~150%之间。结论该方法可用于重组Ⅲ型人源化胶原蛋白冻干纤维中外源性DNA残留量的定量测定。Objective To explore and analyze the residual amount of exogenous DNA in recombinant type Ⅲ humanized collagen freeze-dried fibers by quantitative real-time PCR(qPCR) method, and to determine that this method can be used for DNA quality control feasibility.Methods The host cell residual DNA sample pretreatment kit(magnetic bead method) was used to extract DNA. The E.coli residual DNA detection kit(PCR fluorescent probe method) was used for amplification reaction. The standard curve was drawn, and the qPCR detection method of exogenous DNA residue in recombinant type Ⅲ humanized collagen lyophilized fiber was established.Results Eight batches of recombinant type Ⅲ humanized collagen lyophilized fibers were determined by this method. The residual amount of exogenous DNA was in the range of 0.03-300 pg/μl, with good repeatability, far lower than 10 ng/dose, with good linearity(R^(2)>0.99), and the recovery was between 50%-150%.Conclusion This method can be used for the quantitative determination of exogenous DNA residues in recombinant type Ⅲ humanized collagen lyophilized fibers.

关 键 词：实时荧光定量PCR 重组Ⅲ型人源化胶原蛋白冻干纤维 外源性DNA残留 

分 类 号：R927.1[医药卫生—药学]