蛋白酶消化结合荧光定量PCR法和杂交法检测重组人胰岛素中E._coli细胞DNA残留量的比较

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0.03~2.25×10³ 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 test the E.coli cell DNA residues in recombinant human insulin by using the technology of proteinase K digestion coupled with quantitative polymerase chain reaction(q-PCR),and to make a preliminary validation of the method and compare the method with the slit hybrid-autoradiography method. Methods: The residual host cell DNA in test samples was digested by proteinase K and extracted by nucleic acid purification kits based extraction method,and determinated by Taqman probe based q-PCR,with standard DNA as control. The residual DNA content was analyzed according to the standard curve. The developed method was validated for linear scope,accuracy and precision,and was used for determination of DNA residues in recombinant human insulin. Results: The minimum detection limit of residual E.coli cell DNA by the developed method was 0.03 pg·μL^-1,while the linear range was 0.03-2.25×10³ pg·μL^-1,with a correlation coefficient(r)above 0.98. The recovery rates of spiked samples of various concentrations were approximate,and the relatively standard deviations in 3 tests were also less than 20%. All the residual DNA contents in all batches of hormonal drugs determined by the developed method were less than 10 ng per dose. However,the stripe of 50 pg(the standard DNA)in results determined by hybrid-autoradiography method was not detected. Conclusion: The method of protease digestion coupled with kit elution based extraction solved the technical difficulties in sample pretreatment during residual DNA detection assay successfully. The q-PCR method may replace the hybridization method,and the method is rapid,accurate and sensitive for quantitative determination of residual E.coli cell DNA in recombinant human insulin. And this study will lay the foundation for the future standardization of q-PCR method for detecting DNA residues in drugs.

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