双重实时荧光定量 PCR 检测人维生素 D 受体的方法学构建

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 TaqMan 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 4.00×101～4.00×105 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。

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

Objective To establish a dual real-time fluorescence quantitative polymerase chain reaction (dual realtime 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 amplification efficiency, the intra-assay and inter-assay coefficient of variation were 40 copies/μL, 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/μL, 4.00×101-4.00×105 copies/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.