鸭RLRs和IFN基因SYBRGREENⅠ实时荧光定量PCR检测方法的建立  

Establishment of a Real-time Fluorescence Quantitative PCR Assay for Detection of Duck RIG-I Like Receptors and interferon Genes

作　　者：陈翠腾[1] 傅光华[1] 黄瑜[1] 傅秋玲[1] 万春和[1] 程龙飞[1] 陈红梅[1] 施少华[1] 

机构地区：[1]福建省农业科学院畜牧兽医研究所,福建福州350013

出　　处：《福建农业学报》2015年第3期209-215,共7页Fujian Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31201936);国家现代农业产业技术体系建设专项(CARS-43);福建省科技计划项目--省属公益类科研院所基本科研专项(2011R1025-8)

摘　　要：首次建立以 GAPDH 为内参，同时检测鸭 RIG-I 、MDA5、IFN-α和 IFN-γ等4种 mRNA 相对表达量的SYBR GreenⅠ实时荧光定量 PCR 方法（RT-FQ-PCR）。以目的基因的克隆质粒为标准品，建立标准曲线，并进行熔解曲线、重复性及稳定性分析。该方法 Ct 值线性范围为12．0～32．0；目的基因（RIG-I 、MDA5、IFN-α、IFN-γ）和内参基因（GAPDH）的扩增效率均在95％～105％，其相关系数均在0．99以上；扩增产物的熔解曲线都只出现1个特异性单峰，无引物二聚体或其他非特异性产物生成，RIG-I 、MDA5、IFN-α、IFN-γ和 GAPDH 的 Tm 值依次为（81．9±0．33）、（81．9±0．32）、（93．6±0．23）、（81．8±0．28）、（87．8±0．24）℃，其敏感度均达到100 copies·μL－1，重复性和稳定性好，为从 mRNA 水平上深入研究 RIG-I 样受体、干扰素与鸭机体免疫功能相关性奠定了基础。This study established detection of duck RIG-I ,MDA5,IFN-α and IFN-γ mRNA relative expression levels synchronous of SYBR GreenⅠreal-time quantitative PCR for the first time.GAPDH was used as an internal reference.Cloning plasmids of objective genes were used as the standards for establishing standard curves for analysis of the melting curve,repeatability and stability.The linear range of Ct values of the method was 12.0 to 32.0. The amplification efficiencies of target genes （RIG-I ,MDA5,IFN-α,IFN-γ）and the reference gene （GAPDH ）were from 95% to 105%,and the correlation coefficients were above 0.99. The melting curve of amplification product only appeared a specific single peak,and there was no primer dimer or other non-specific product formation.Tm values of RIG-I ,MDA5,IFN-α,IFN-γ and GAPDH were （81.9±0.33）,（81.9±0.32）, （93.6±0.23）,（81.8±0.28）and （87.8 ±0.24）℃,respectively.The sensitivity reached 100 copies·μL-1 ,and repeatability and stability were good.This method would lay the foundation for further study of correlation between duck RIG-I like receptor,interferon and immune function from the mRNA levels.

关 键 词：RIG-I 样受体 干扰素 实时荧光定量 PCR 

分 类 号：S852.4[农业科学—基础兽医学]