【摘要】 根据GenBank上鸡α-、β-、γ-干扰素(ChIFN-α、-β、-γ)基因的序列,在保守区设计并合成各自特异引物,并以鸡3-磷酸甘油脱氢酶(GAPDH)基因为内参,采用SYBR GreenⅠ染料以建立实时荧光定量PCR检测方法。将IFN-α、-β、-γ基因克隆至pGEM-T载体上,以各阳性质粒作为标准品,构建标准曲线,并进行了熔解曲线分析。结果表明,鸡IFN-α、-β、-γ和GAPDH基因的Ct值与标准品稀释度在1×102~1×108copies/μL范围分别呈良好的线性关系,r2均大于0.990。熔解曲线分析表明,产物为特异的单峰,检测周期从RNA提取到荧光定量PCR结束只需4 h。建立的鸡IFN-α、-β、-γ基因实时荧光定量PCR灵敏度高、特异性强、检测周期短,为在mRNA水平对鸡IFN的定量分析奠定了基础。更多还原

【Abstract】 According to the chicken’s IFN-α,-β,and-γ(ChIFN-α,-β,-γ) gene sequences available in GenBank,five pairs of primers were designed for developing a SYBR GreenⅠ quantitative real-time PCR method to detection IFN-α,-β,and-γ genes of chicken while the chicken glyceraldehyde-3-phosphate dehydrogenase(ChGAPDH) gene was used as an internal control.To establish the standard curve,the positive plasmid of each cytokine served as a standard.The melting curve was also analyzed.The results showed that the Ct of ChIFN-α,-β,and-γ or ChGAPDH genes had a good linear relationship(r2>0.990) with the standard samples from 1×102to 1×108 copies/μL,and the melting curve showed a single peak.The deve-loped real-time PCR assay could quickly detect IFN-α,-β,and-γ genes in expansion range with high efficiency,thus providing the basis for quantitative analysis of IFN-α,-β,and-γ gene expression.更多还原