SYBR-Green实时定量PCR用于Vero宿主细胞DNA残留量的检测

Detection of residual Vero cell DNA by SYBR-Green real-time PCR

目的:建立基于SYBR-Green荧光染料实时定量PCR检测Vero宿主细胞DNA残留量的方法。方法:提取Vero细胞基因组DNA,设计细胞基因组高度重复顺序AGMr(HindIII)-1基因片段的特异性引物,通过PCR扩增AGMr(HindIII)-1序列中一段特异性cDNA片段,克隆至pGEM-T Vector,重组质粒经酶切及测序鉴定合格后命名为pGEM-T/AGMr(HindIII)-1-S。结果:使用重组质粒和细胞基因组DNA分别作为检测标准品,均取得了良好的结果,其检测灵敏度分别达到了0.03 fg.μL-1和0.03 pg.μL-1。结论:SYBR-Green实时定量PCR可用于Vero宿主细胞DNA残留量的准确定量。

Objective： To establish SYBR-Green real-time PCR to detect the residual Veto cell DNA. Methods： Extracted Vero cell genomic DNA; designed the long tandem repeat sequence（LTR） gene specific primer and obtained the cDNA of AGMr（ HindⅢ）-I fragment. Clone the long tandem repeat sequence（LTR） of genornic DNA of Vero cells to pGEM-T Vector,insert-positive clones of pGEM-T/AGMr（HindⅢ）-1-S were obtained by re- striction analysis and sequencing. Results： Good results were obtained when using pGEM-T/AGMr（ HindⅢ）-1-S and Vero cell genomic DNA as standard Respectively. The sensitivity of the developed quantitative PCR method was 0.3 fg and 300 fg respectively. Conclusion： SYBR-Green real-time PCR method might be used for quantitation of residual Vero cell DNA in vaccine.