中文摘要：

为建立猪IFN-β及IRF-3基因实时荧光定量PCR检测方法,依据GenBank中IFN-β和IRF-3基因的保守序列,设计并合成各自特异性引物,并以β-actin为内参基因,采用SYBR Green-Ⅰ为染料,建立实时荧光定量检测方法。提取猪肺泡巨噬细胞总RNA,经反转录得cDNA,用特异性引物经PCR扩增得到IFN-β和IRF-3基因,将其克隆至pMD-19T载体,经测序鉴定后得重组质粒,依次10倍稀释做为标准品,建立标准曲线及溶解曲线。结果表明,β-actin基因、IFN-β基因和IRF-3基因标准曲线线性关系较好,R2≥0.997;溶解曲线为特异性单峰,无非特异性扩增,检测下限可达100个拷贝/μL。建立的猪IFN-β和IRF-3基因实时荧光定量RT-PCR检测方法,特异性强、重复性好,为从分子水平上研究猪的免疫应答奠定了基础。

英文摘要：

To establish real-time fluorescent quantitative RT-PCR assays for detecting porcine IFN-β and IRF-3,sevral specific primer pairs were designed according to the porcine＇s IFN-β and IRF-3 gene sequences available in GenBank,and the porcine β-actin gene was used as an internal gene control.The total RNA was extracted from porcine alveolar macrophages.The reverse transcriptase PCR was used to obtain the first strand cDNA.Fragments for IFN-β and IRF-3 were amplified by PCR from the synthesized cDNA using the designed specific primers.The PCR products were purified and cloned into pMD-19T vector.The positive recombined plasmids were serially diluted and used as a standard.The standard and melting curve was analyzed.The results showed that the Ct value of β-actin,IFN-β and IRF-3 genes had a good linear relationship（R2≥0.997） and the melting curve showed a single peak.The established real-time PCR methods can detect 100 copies of IFN-β and IRF-3 mRNA.The developed real-time PCR using SYBR GreenⅠdye had high sensitivity,sepcifity and reproductivity,and could used as an effective tool for detection and quantification of IFN-β and IRF-3.