DNA聚合酶中宿主细胞核酸残留的分析

目的分析市售不同厂家DNA聚合酶中的宿主细胞核酸残留。方法根据E.coli 16S rRNA基因序列设计特异引物及探针,建立基于Taqman探针技术的Real-time PCR检测方法,检测基因工程制品中E.coli的核酸残留。结果建立的Real-time PCR法检测市售不同厂家的DNA聚合酶中均有宿主细胞E.coli核酸残留,但不同来源的DNA聚合酶其宿主细胞核酸残留量不同。结论应用PCR方法检测基因工程产品,尤其是E.coli制备的生物制品时,应注意DNA聚合酶中核酸残留对检测结果的影响;应用Real time PCR法定量检测时,建议将Ct值控制在30以内,以减小DNA聚合酶背景残留物的影响。

Analysis of residual host cell nucleic acid in DNA polymerase

Objective To analyze the residual host cell nucleic acid in commercial DNA polymerase products manufactured by various manufacturers.Methods A Taqman probe-based real-time PCR method was developed by using the specific primers and probes designed according to E.coli 16S rRNA gene sequence,and used for analysis of residual E.coli DNA in recombinant products.Results Residual E.coli DNAs were detected in all the DNA polymerase products manufactured by various manufacturers by the developed real-time PCR,of which the contents were different.Conclusion When recombinant products especially those prepared with E.coli were tested by PCR,the effect of residual host cell nucleic acid in DNA polymerase on test result should be paid more attention.It suggested that the Ct value of real-time PCR for quantitative determination should be controlled to 30 or below so as to minimize the effect of background residue of DNA polymerase.