猪源凋亡相关基因SYBR Green Ⅰ荧光定量RT-PCR方法的建立

Development of real-time RT-PCR assay based on SYBR Green Ⅰ for detection of porcine apoptosis-related cytokines

为检测高致病性猪繁殖与呼吸综合征病毒(PRRSV)感染的猪胸腺中凋亡相关因子的变化,本研究针对猪TNF-α、TNFR1、FasL、Fas、Bax、Bcl-2、caspase-3基因及内参β-actin基因设计特异性引物,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测TNF-α、TNFR1、FasL、Fas、Bax、Bcl-2、caspase-3基因的SYBR Green I qRT-PCR方法。结果显示,该方法线性关系好(R2≥0.998),敏感性高,初始模板的检出下限为10拷贝/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,批内、批间重复试验的变异系数均小于3%,具有良好的重复性。本研究为细胞凋亡相关因子变化趋势的研究提供方法。

For detection of the related apoptotic cytokine changes in the atrophic thymus of piglets induced by porcine reproductive and respiratory syndrome virus （PRRSV） infection. The recombinant plasmids containing the target genes were constructed as a standard control for different cytokines and [3-actin, a set of SYBR Green I real time RT-PCR assays were developed for TNF-et, TNFR1, FasL, Fas, Bax, Bcl-2 and caspase-3 detections with the specific primers designed based on the genes of the related cytokines in GenBank. The results showed that these methods had a single specific melting peak at different Tm value for each cytokine and all showed a linear dynamic, with the correlation coefficient R2 of 0.998, and they were sensitivity and repeatability, the detection limit were 10 copies/uL, the coefficient of variation were lower than 3%. This study provided a method for the detection of apoptosis related cytokines.