CD63基因TaqMan实时荧光定量PCR检测方法的建立及评价

Development and evaluation of a real-time TaqMan reverse transcription-PCR assay for quantification of CD63 gene

为建立一种CD63基因TaqMan实时荧光定量PCR检测方法,以期为CD63基因的动态检测提供有效的实验手段,根据GenBank中公布的CD63基因序列,设计1对荧光定量PCR引物及1条探针,建立一种快速检测CD63基因的实时荧光定量PCR方法,并对建立的方法进行条件优化。结果显示,建立的实时荧光定量PCR方法线性关系较好,以质粒标准品构建的标准曲线相关系数r2为0.997;灵敏性及重复性均较高;应用建立的方法对多种种属来源细胞进行检测,能检出不同类型细胞中CD63的含量。结果表明,成功建立了一种快速、准确检测CD63基因的TaqMan实时荧光定量PCR方法。

To develope a real-time TaqMan reverse transcription-PCR（RT-PCR） assay for effective quantification of CD63 gene, based on the CD63 gene sequence retrieved from Gen Bank, a pair of PCR primers and a probe were designed. Then a real-time Taq Man RT-PCR assay for accurate quantification of CD63 gene was developed and optimized. In result, the real-time Taq Man RT-PCR assay had a good linear relationship. The standard curve correlation coefficient r2 was 0.997. It also had a high sensitivity and reproducibility.We successfully detected and quantified CD63 gene in cells from different species. In conclusion, a rapid and accurate method for quantification of CD63 gene by real-time Taq Man reverse transcription-PCR was successfully developed.