牛IFN-α、IFN-β及IFN-γ mRNA实时SYBR GreenⅠ定量RT-PCR检测方法的建立及应用  

Establishment of a SYBR GreenⅠreal-time quantitative RT-PCR assay for detection of bovine alpha-interferon,bata-interferon and gamma-interferon mRNA

作　　者：韩猛立[1,2] 杨井泉[1,2] 姚守秀[2] 黄新[1] 薄新文[1] 钟发刚[1] 

机构地区：[1]新疆兵团绵羊繁育生物技术重点实验室,新疆石河子832000 [2]石河子大学动物科技学院,新疆石河子832003

出　　处：《中国预防兽医学报》2010年第10期762-767,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金："973"计划前期研究专项(2008CB117017);国家自然基金项目(30960286);兵团博士资金项目(2007JC15)

摘　　要：为建立灵敏度高、特异性强、能够快速检测牛α-、β-、γ-干扰素(BoIFN-α、BoIFN-β、BoIFN-γ)mRNA的实时荧光定量PCR方法,本研究根据GenBank中登录的BoIFN-α、BoIFN-β和BoIFN-γ基因序列,分别在其保守区内设计并合成特异引物,以牛3-磷酸甘油脱氢酶(GAPDH)mRNA为内参,建立SYBR GreenⅠ实时荧光定量PCR检测方法。将BoIFN-α、BoIFN-β和BoIFN-γcDNA分别克隆到pMD18-T载体中,转化大肠杆菌DH5α内,经PCR及测序鉴定,将得到的阳性重组质粒制备成标准品,建立SYBR Green荧光定量PCR标准曲线并分析溶解曲线,进行灵敏性、特异性和重复性试验。结果表明:BoIFN-α、BoIFN-β、BoIFN-γ和GAPDH基因的Ct值与标准品稀释度在1×101copies/μL～1×107copies/μL内均呈良好的线性关系,相关系数均大于0.991。熔解曲线分析表明,产物为特异的单峰,特异性和重复性较好。本研究建立的检测牛IFN-α、IFN-β和IFN-γmRNA的实时荧光定量PCR方法,为牛IFN mRNA水平上的定量分析奠定了基础。This study aimed to develop a SYBR Green Ⅰ real-time quantitative RT-PCR assay for rapid detection of bovine IFN-α,IFN-β and IFN-γ（BoIFN-α,-β and-γ） mRNA.The assay was established using four pairs of specific primers designed according to the BoIFN-α,-β and-γ gene sequences,with the bovine glyceraldehyde-3-phosphate dehydrogenase（BoGAPDH） mRNA as an internal control.The target genes were amplified by conventioanl RT-PCR,and used as templates for generating standard curve and melt curve.The results showed that the Ct value of BoIFN-α,-β,-γ and BoGAPDH mRNA had a good linear relationship（r20.991） with the standard from 10 copies/μL to 107 copies/μL,and the melting curve showed a single peak.The reproducibility,specificity and sensitivity of the assay were also higher than conventional RT-PCR in detecting respective target mRNA.Therefore,the real-time quantitative PCR assay could be effective method for quantitative analysis of IFN-α,-β and-γ gene expression.

关 键 词：SYBR GreenⅠ 实时荧光定量PCR 牛 α-干扰素 Β-干扰素 γ-干扰素 

分 类 号：S852.4[农业科学—基础兽医学]