牛IFN-α、IFN-β荧光定量PCR检测方法的建立

Establishment of Fluorescence quantitative PCR for detection of Bovine IFN-αand IFN-β

建立一种检测牛IFN-α、IFN-β基因SYBY GreenⅠ实时荧光定量PCR方法,旨在为抗病毒相关研究奠定基础。设计合成扩增牛IFN-α、IFN-β基因的特异性引物,采用RT-PCR从牛脾脏中扩增出目的DNA片段,将其克隆至pMD18-T载体获得牛IFN-α、IFN-β质粒标准品,以质粒标准品为模板,优化荧光定量PCR反应条件,建立牛IFN-α、IFN-β基因SYBY GreenⅠ实时荧光定量PCR方法,结果表明,牛IFN-α、IFN-β标准曲线的R2分别为0.9588和0.99652,线性良好;扩增效率为0.97和0.91,效率高;熔解曲线均为单峰,特异性较高,并具有良好的重复。结果证实,本研究所建立的方法敏感性高,特异性强,重复性好。

A SYBY GreenⅠreal-time fluorescence quantitative PCR method for detection of bovine IFN-α,IFN-βgene was established in order to lay a foundation for antiviral research.The specific primers for amplification of bovine IFN-α,IFN-βgene were designed and synthesized.The target DNA fragment was amplified from bovine spleen by RT-PCR and inserted to pMD18-T vector to obtain bovine IFN-α,IFN-βplasmid standard.Using plasmid standard as template,the fluorescence quantitative PCR reaction conditions were optimized,and the SYBY GreenⅠreal time fluorescence determination of bovine IFN-α,IFN-βgene was established.The results showed that the R2 of bovine IFN-αand IFN-βstandard curves were 0.9588 and 0.99952,respectively,which were linear;the amplification efficiency was 0.97 and 0.91,the efficiency was high;and the melting curves were single peak,the specificity was high and the repetition was good.The results showed that the method established in this study had high sensitivity,specificity and reproducibility.