质粒DNA中宿主菌基因组DNA残留检测方法的比较

目的对检测质粒DNA中宿主菌基因组DNA含量的Southern blot和Real-time PCR法进行比较。方法PCR扩增宿主菌染色体DNA的16SrRNA片段,以地高辛标记回收的目的片段为探针,对提取的质粒pcDNAH进行Southern blot,同时根据16SrRNA基因序列设计探针,建立Real-time PCR反应体系和标准曲线,分别检测质粒中宿主菌基因组DNA的含量。结果应用Southern blot和Real-time PCR检测质粒pcDNAH中宿主菌基因组DNA的含量,结果均符合WHO相关标准。结论两种检测方法各有优缺点,均可用于检测质粒DNA中宿主菌基因组DNA的残留,可根据不同情况,采用不同方法或配套使用。

Comparison of Two Methods for Determination of Residual Host Genomic DNA in Plasmid DNA

Objective To compare two methods,i.e.Southern blot and real-time PCR,for determination of residual genomic DNA in plasmid DNA.Methods The 16S rRNA fragment of host E.coli chromosome was amplified by PCR,labeled with digoxin and used as a probe for Southern blotting with plasmid pcDNAH extracted from recombinant E.coli DH5α.Meanwhile,a probe was designed according to the gene sequence of 16S rRNA and used for determination of host genomic DNA by real-time PCR,based on which a standard curve was plotted.Results Both the residual host genomic DNA contents in plasmid pcDNAH determined by Southern blot and real-time PCR met the relevant WHO requirements.Conclusion Both Southern blot and real-time PCR were suit-able for the determination of residual host genomic DNA in plamid DNA,which may be applied alone or in combination.