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目的:利用蛋白酶K消化结合荧光定量聚合酶链式反应(q-PCR)技术,检测重组人胰岛素原料中E.coli细胞DNA残留量,并进行初步的方法学验证,与狭缝杂交-放射自显影方法进行比较。方法:通过蛋白酶K消化样品,利用核酸纯化提取试剂盒提取DNA,然后采用Taqman探针法对样品和标准DNA进行定量PCR测定,再根据标准曲线对样品中DNA残留量进行分析。对方法进行线性、范围、准确度和精密度的验证,对重组人胰岛素中DNA残留量进行测定。结果:该方法检测E.coli细胞DNA残留的最低定量限度为0.03 pg·μL~(-1),DNA含量在0.03~2.25×10~3 pg·μL~(-1)范围内线性关系良好,相关系数在0.98以上;该方法检测不同加标样品回收率较接近,3次测定的相对标准偏差均小于20%;该方法检测各批次重组人胰岛素的DNA残留量均小于10 ng·剂量~(-1),而狭缝杂交——放射自显影法50 pg(标准DNA)均未出现条带。结论:蛋白酶消化结合试剂盒洗脱解决了残留DNA检测中样品前处理的难题,定量PCR检测可能代替杂交法,快速、准确、灵敏地对重组人胰岛素原料中E.coli细胞残余DNA进行定量测定,为将来荧光定量PCR检测药物中宿主DNA残留方法标准化奠定了基础。
OBJECTIVE: To detect the DNA residues in E.coli cells of recombinant human insulin by protease K digestion and q-PCR and perform preliminary methodological verification. Development method for comparison. Methods: The samples were digested with proteinase K, extracted with DNA purification kit, and then quantitatively determined by Taqman probe method. Then the DNA residues in samples were analyzed by standard curve. The method was validated for linearity, range, accuracy, and precision, and the amount of DNA remaining in recombinant human insulin was determined. Results: The minimum limit of quantitation (DNA) of E.coli cells was 0.03 pg · μL ~ (-1) and the DNA content was within the range of 0.03 ~ 2.25 × 10 ~ 3 pg · μL ~ (-1) The correlation coefficient was above 0.98. The recoveries of different spiked samples by this method were close, and the relative standard deviations of three determinations were less than 20%. The DNA residues of recombinant human insulin in each batch were less than 10 ng · dose- (-1), while no band was observed in 50 pg (standard DNA) by slit hybridization-autoradiography. Conclusion: The protease digestion combined with kit elution can solve the problem of sample pretreatment in residual DNA detection. Quantitative PCR detection may replace the hybridization method to quantitatively determine the residual DNA of E.coli cells in recombinant human insulin rapidly, accurately and sensitively , Which laid the foundation for the future standardization of fluorescence quantitative PCR in detecting host DNA residues in drugs.