双重实时荧光定量PCR检测人维生素D受体的方法学构建

Establishing a method for detection of human vitamin D receptor using dual real-time fluorescence quantitative PCR

目的 建立单管检测人维生素D受体（VDR）及甘油醛-3-磷酸脱氢酶（GAPDH）基因的双重实时荧光定量聚合酶链反应（dual real-time PCR）的方法。方法 以GAPDH基因为内参,采用Primer Premier 5.0软件设计特异性引物及Taq Man探针,进行PCR扩增检测VDR基因。将VDR及GAPDH扩增产物片段纯化后克隆构建成重组质粒,作为定量检测基因表达的标准品,并用于分析该方法的灵敏度和重复性。结果 PCR扩增产物经测序分析证实为VDR及GAPDH特异性片段;该方法检测VDR与GAPDH灵敏度达40拷贝/μL;线性范围为4.00×10~1~4.00×10~5拷贝/μL;决定系数R2分别为0.998、0.999;扩增效率E分别为96.10%、85.15%;批内变异系数（CV）分别为0.09%~1.21%、0.35%~0.88%;批间CV分别为0.17%~0.51%、0.51%~2.46%。结论 成功建立了单管检测人VDR及GAPDH的双重实时荧光定量PCR方法,且该方法特异性好、灵敏度高、可快速高通量检测VDR的相对表达量,有效缩短时间,减小实验误差。

Objective To establish a dual real-time fluorescence quantitative polymerase chain reaction （dual real- time PCR） assay to detect human vitamin D receptor （VDR） and glyceraldehyde- 3- phosphate dehydrogenase （GAPDH）. Methods GAPDH gene was used as the internal control. The specific primers and TaqMan probes were designed by Primer Premier 5.0 software, which were applied to detect the VDR/GAPDH mRNA levels. The obtained PCR products were purified to construct the VDR/GAPDH recombinant plasmid, which was taken as the standard to analyze the sensitivity and repeatability of the method. Results The amplification products were confirmed as the specific fragment of VDR/GAPDH by DNA sequencing instrument. The results showed that the sensitivity, linear range, the determinate coefficient, the amplifica- tion efficiency, the intra-assay and inter-assay coefficient of variation were 40 copies/p,L, 4.00× 101-4.00× 105 copies/μL, 0.998, 96.10%, 0.09%-1.21%, 0.17%-0.51% for VDR, and 40 copies/p,L, 4.00× 101-4.00× 10s eopies/μL, 0.999, 85.15%, 0.35%-0.88%, 0.51%-2.46% for GAPDH, respectively. Conclusion These results demonstrate that the dual real-time PCR assay with high sensitivity and specificity can detect the relative expressions of human VDR by single reaction tube, which can effectively shorten the time and reduce the experimental error.