实时荧光PCR检测鸡GAPDH基因方法的建立

Establishment of Real-time Fluorescence PCR for GAPDH Gene of Chicken

根据GenBank上鸡的3-磷酸甘油醛脱氢酶(glyceraldehyde-3-phosphate dehy-drogenase,GAPDH)基因的序列,在保守区域设计并合成一对引物,采用SYBR GreenⅠ染料建立了荧光定量PCR法。采用梯度浓度的GAPDH基因重组标准品质粒DNA进行荧光定量PCR并建立了标准曲线,经分析显示标准曲线的Ct值与标准品浓度的对数值之间存在良好的线性关系;动力学曲线分析表明,在本研究所建立的反应体系和反应条件下,标准曲线的灵敏度都达到10拷贝/反应。实时荧光定量PCR检测GAPDH基因表达水平标准品质粒和标准曲线的建立,为GAPDH基因作为内参基因进行鸡功能基因与病原基因表达的定量分析奠定了基础。

According to the chicken GAPDH gene sequences available in GenBank,a pair of primers was designed for the establishment of a SYBR Green Ⅰ quantitative real-time PCR method for GAPDH gene of chicken. GAPDH was amplified by real-time fluorescence quantitative PCR from the plasmid DNA which was diluted to series standard concentrations,and the standard curves were established which indicate that there is a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen. Under the established condition the reaction,the sensitivity of the methods was 10 copies/μL by the analysis of the dynamic curve. The recombinant plasmid and standard curve for GAPDH real-time quantitative PCR were constructed successfully. It provided the basis for GAPDH gene of chicken as a reference gene in quantitative analysis of mRNA expression.