重组E.coli宿主中质粒DNA拷贝数qPCR检测方法的建立及验证

Development and verification of qPCR method for detection of recombinant E.coli plasmid DNA copy number in host

目的建立重组E.coli宿主中质粒DNA(pDNA)拷贝数的qPCR检测方法,并进行验证,以期为mRNA疫苗研发提供可靠的检测方法。方法根据E.coli Top10基因组DNA序列设计用于qPCR扩增的引物及探针,并优化引物浓度。提取重组E.coli/pUC57-HA的全DNA(宿主DNA+pDNA),以其为模板,建立pDNA拷贝数的qPCR检测方法,并验证方法的线性范围、特异性及重复性。将重组E.coli/pUC57-HA于37℃培养24 h,每2 h取样,采用建立的方法检测pDNA拷贝数。结果确定最佳引物(F/R)浓度均为0.5μmol/L。重组E.coli/pUC57-HA全DNA在10~1~10~5倍稀释范围内,与Ct呈良好的线性关系,R^(2)均=1.00;E.coli/pUC57-HA及E.coli Top10的扩增结果为阳性,阴性对照(ddH_(2)O)未见扩增曲线;3次重复检测重组E.coli/pUC57-HA的pDNA拷贝数CV<5%。E.coli/pUC57-HA培养0~6 h期间pDNA拷贝数呈平稳趋势,6~12 h期间呈下降趋势,12~16 h期间呈增长趋势,16~24 h又趋于平稳。结论建立的qPCR法具有良好的特异性及重复性,可用于重组E.coli宿主中pDNA拷贝数的检测,有效监测菌株培养过程中pDNA含量变化。

Objective To develop and verify a detection method for recombinant E.coli plasmid DNA(pDNA)copy number in the host based on qPCR technology platform,so as to provide a reliable detection method for the development of mRNA vaccine.Methods According to the Top10 genomic DNA sequences of E.coli,the primers and probes were designed for qPCR amplification,and the primer concentration was optimized.The whole DNA(host DNA+pDNA)of recombinant E.coli/pUC57-HA was extracted and used as the template to develop a qPCR method for the detection of pDNA copy number.The linear range,specificity and reproducibility of the method were verified.The recombinant E.coli/pUC57-HA was cultured at 37℃for 24 h,and the samples were taken every 2 h and detected for the pDNA copy number by the developed method.Results The optimum primer concentration(F/R)was 0.5μmo/L.The whole DNA of recombinant E.coli/pUC57-HA showed a good linear relationship with Ct values in the dilution range of 101-105 times,each R2=1.00.The amplification resultsof E.coli/pUC57-HA and E.coli Top10 were positive,and the negative control(ddH2O)showed no amplification curve.The CV of three repeated detections for pDNA copy number of recombinant E.coli/pUC57-HA was less than 5%.The pDNA copy number of E.coli/pUC57-HA was stable during the incubation period of 0-6 h,decreased during 6-12 h,increased during 12-16 h,and stabilized again during 16-24 h.Conclusion The developed qPCR method has good specificity and reproducibility,which can be used to detect the copy number of pDNA in recombinant E.coli host,and effectively monitor the change of pDNA content during culture.