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