茎环RT-PCR检测马立克氏病毒感染鸡相关miRNA方法的建立  

Detection of MiRNA Levels in Chicken Artificially Infected with Marek′s Disease Virus by Stem-loop Real-time Quantitative PCR

作　　者：陈燕梅[1] 连玲[1] 曲鲁江[1] 刘长军[2] 杨宁[1] 

机构地区：[1]中国农业大学动物科技学院,北京100193 [2]中国农业科学院哈尔滨兽医研究所,黑龙江哈尔滨150001

出　　处：《中国家禽》2011年第9期9-13,共5页China Poultry

基　　金：现代农业产业技术体系建设专项资金(CARS-41);家禽产业技术体系北京市创新团队专项资金;"十二五"国家科技支撑项目(2011BAD28B03)

摘　　要：本研究旨在建立一种检测马立克氏病毒人工感染鸡相关miRNA表达变化的RT-PCR方法,探讨该方法在miRNA定量检测方面的应用价值。试验选择6个具有典型肿瘤结节的马立克氏病毒感染脾脏样品作为研究材料,同时采用6个正常鸡的脾脏样品作为对照材料。利用特异的茎环反转录引物以及miRNA上下游引物对gga-miR-181a进行基于SYBR GreenⅠ的荧光定量PCR检测,并以U6为内参基因、2-ΔΔCt法检测gga-miR-181a的相对表达量。结果,在马立克氏病毒人工感染鸡的脾脏和正常鸡的脾脏中成功检测出gga-miR-181a-1的表达。证明茎环RT-PCR法能用于检测马立克氏病毒人工感染鸡的相关miRNA,并具有准确、快速、节约成本的优点。In this study,stemp-loop RT-PCR method based on SYBR Green I for detecting miRNA in chicken artificially infected with Marek＇s disease virus （MDV） was established,and related application value of this method in quantitative assay miRNA was investigated. Six phymatoid spleen samples from MDV-infected chickens and six normal spleens from uninfected chickens were used as samples. After cloning （with pMD19-T plasmid and DH5c~） and sequencing for eonfirmatiorl,stem-loop RT primers and miRNA specific quantitative PCR forward primers and universal reversed primers for gga-miR-181a-1 and U6 endogenous control gene,were used to detect the relative expression levels of gga-miR-181a-1 using 2. In conclusion, stern-loop RT-PCR was a sensitive and fast method which could be used to detect expression levels of miRNA in chicken artificially infected with MDV.

关 键 词：鸡 马立克氏病毒 MIRNA 茎环RT-PCR 

分 类 号：S662.5[农业科学—果树学]