鸡IFN-α和IFN-β及IFN-γ基因实时荧光定量RT-PCR检测方法的建立  

Establishment of a real-time fluorescent quantitative RT-PCR assay for detection of chicken IFN-α,IFN-β and IFN-γ genes

作　　者：张贺楠[1] 赖汉漳[1] 齐岩[1] 孔留五[1] 张小桃[1] 曹伟胜[1] 廖明[1] 

机构地区：[1]华南农业大学兽医学院农业部动物重大疫病防控重点开放实验室,广东广州510642

出　　处：《中国兽医科学》2009年第2期173-177,共5页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30771612)

摘　　要：根据GenBank上鸡α-、β-、γ-干扰素（ChIFNα-、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SYBRGreen工染料以建立实时荧光定量PCR检测方法。将IFNα-、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IFNα-、β-、γ-和GAPDH基因的C1值与标准品稀释度在1×10^2～1×10^8 copies／μL范围分别呈良好的线性关系，r^2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4h。建立的鸡IFNα-、β-、γ-基N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。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 Green I quantitative real-time PCR method to detection IFN-α,-β, and-γ genes of chicken while the chicken glyeeraldehyde-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 ChlFN-α,-β, and-γ or ChGAPDH genes had a good linear relationship（r^2 〉0. 990） with the standard samples from 1×10^2to 1 × 10^8 copies/μL,and the melting curve showed a single peak. The developed 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, IFN-β and IFN-γ gene expression.

关 键 词：实时荧光定量PCR 鸡 Α-干扰素 Β-干扰素 γ-干扰素 

分 类 号：Q511[生物学—生物化学] Q503