实时荧光定量PCR检测人apoM方法的构建

Establishing a method for quantitation of apolipoprotein M load using real-time fluorescence quantitative-PCR

目的建立检测人载脂蛋白M（apoM）的实时荧光定量PCR技术。方法采用Pri merExpress Versions2.0设计引物及Taq Man探针,检测apo M基因。将扩增的apo M基因片段纯化后与pMD19-T载体连接,克隆构建含apo M基因片段的重组质粒,作为定量检测apo M基因表达的标准品。结果PCR扩增产物经测序分析证实为apo M特异性片段。本法灵敏度为40copies/μl,线性范围为4.00×101~4.00×108copies/μl,相关系数为1.000,扩增效率E为90.5%,批间变异系数为6.38%~17.9%。结论成功建立了检测人apo M的实时荧光定量PCR方法,且该方法特异性好,灵敏度高。

Objective To develop a method for quantitation of apolipoprotein M（apoM）mRNA load using real-time fluorescence quantitative-PCR.Methods The primers and TaqMan probe targeted at signature and conserved sequence of apoM gene were designed by software ＂Primer Express Versions 2.0＂ and applied to detect the apoM mRNA levels.The apoM recombined plasmid was constructed by conventional molecular biological techniques,which was taken as the standard of apoM quantitative detection.The reaction system was optimized,and the sensitivity,specificity and reproducibility were evaluated.Results The amplification products were confermed as the specific fragment of apoM by DNA sequencing instrument.The results showed that the sensitivity,linear rang,the interassay coefficient of variation,the correlation coefficient of this assay were 4×101copies/μl,4.00×101～4.00×108 copies/μl,6.38%-17.9%,1.00,respectively.Conclusion The method for quantitation of apolipoprotein M（apoM）mRNA load has been established successfully with high sensitivity,specificity and stability and can be applied to quantify the apoM mRNA levels.