实时定量PCR检测重组技术产品中宿主基因组DNA残留___

目的：建立Real-time PCR方法，用于定量检测重组技术产品中宿主基因组DNA的残留。方法：以ABI 7500 FAST平台为基础，选择大肠杆菌23S核糖体RNA基因为靶标基因设计扩增引物，建立基于SYBR Green I荧光染料的real-time PCR检测方法，用于重组技术产品宿主DNA残留的质量控制。结果：该法检测宿主DNA残留灵敏度可达到1 fg（1×10-6 ng），DNA浓度在1×10-5～100 ng•μL-1范围内线性良好，其标准曲线的相关系数为0.99以上。应用该法对4批重组人粒细胞刺激因子进行测定，外源DNA残留量分别为每剂量6.699×10-3，7.948×10-3，9.362×10-3，7.506×10-3 ng。结论：该方法具有操作简便、灵敏度高等优点，可用于重组产品中大肠杆菌残余DNA的定量测定。

Objective:To develop a real-time PCR for determination of residual host genomic DNA in recombinant products.Methods:Based on ABI 7500 FAST system,amplify residual host genomic DNA using designed primers target to E.coli 23S ribosome RNA gene and SYBR Green I dye and determine the real-time fluorescence intensity of amplified product by the developed real-time PCR.Results:The developed real-time PCR had high sensitivity up to 1 fg (1×10-6 ng) of DNA with good linearity (r ≥0.99) in the concentration range of 1×10-5-100 ng•μL-1.Four batches of recombinant human G-CSF products were tested.The residual DNA was 6.699×10-3,7.948×10-3,9.362×10-3 and 7.506×10-3 ng per dose,respectively.Conclusion:This method is convenient and sensitive,and can be used in quantitative test for E.coli residual DNA in recombinant products.