2种定量PCR法与探针杂交法在检测重组白介素1受体拮抗剂中宿主DNA残留量的比较

目的：建立Taqman探针技术的定量PCR方法，对大肠杆菌E.coli/BL21菌株生产的重组白介素1受体拮抗剂（rhIL-1ra）中的残留宿主DNA进行了定量分析，并与SYBR Green法和地高辛探针杂交法进行比较。方法：以BioRad Chromo 4荧光定量PCR仪为平台，选择大肠杆菌23S核糖体RNA基因为靶基因，设计引物和Taqman探针，以纯化的宿主基因组DNA为标准品，对供试品中残留DNA进行定量。同时在重复性，精密度，检测限等方面与SYBR Green方法和地高辛探针杂交法进行比较。结果：本实验建立的Taqman探针法检测限为0.01 ng·mL^-1，在0.01～1000 ng·mL^-1区间同样具有良好的线性（r=0.997），回收率89.7%～136.0%。测定4批次供试品中残留DNA浓度为0.052、0.092、0.096、0.025 ng·mL^-1。结论：Taqman探针法与SYBR Green法检测灵敏度均达到0.01 ng·mL^-1，在重复性与精密度方面效果一致，且均优于探针杂交方法。

Objective: To develop a Taqman probe-based quantitative PCR method for the determination of residual host genomic DNA in E.coli/BL21 recombinant human interleukin-1 antagonist(rhIL-1Ra),and to compare the efficiency of the method with that of SYBR Green PCR method and digoxin (DIG) probe hybridization method. Methods: Based on BioRad Chromo 4 system,PCR primer and Taqman probe were designed to amplify 23S ribosomal RNA of E.coli.Residual host DNA concentration was calculated by simple linear regression with standard host genomic DNA sample.Repeatability,efficiency and limit of detection of this method were compared with that of SYBR Green method and DIG probe hybridization method. Results: The limit of detection of the Taqman probe PCR method for E.coli genomic was 0.01 ng · mL^-1,and this method showed a good linearity within the test range of 0.01-1000 ng·mL^-1(r=0.997) and the recovery was between 89.7%-132%.The residual DNA concentrations of 4 tested product batches were 0.052,0.092,0.096,0.025 ng· mL^-1 respectively. Conclusion: The Taqman method and the SYBR Green method both have high sensitivity of 0.01 ng·mL^-1 and high repeatability and accuracy,and significantly outperform the probe hybridization method.

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