猪IFN-γ mRNA TaqMan荧光定量RT-PCR检测方法的建立  

Development of a TaqMan fluorescent quantitative RT-PCR assay for detection of porcine IFN-γ mRNA

作　　者：韦天超[1,2] 田志军[1] 周艳君[3] 安同庆[1] 肖燕[1] 彭金美[1] 姜一峰[1] 童光志[1,3] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室猪传染病研究室,黑龙江哈尔滨150001 [2]广西大学动物科学技术学院,广西南宁530005 [3]中国农业科学院上海兽医研究所,上海200232

出　　处：《中国兽医科学》2009年第2期168-172,共5页Chinese Veterinary Science

基　　金：国家重点基础研究发展规划(973)项目(2005CB523200)

摘　　要：为建立一种检测猪γ-干扰素（IFN-γ）的荧光定量RT—PCR方法，针对猪IFN-γ基因和管家基因cyclophilin A（CyPA）的核苷酸序列分别设计了特异性引物和TaqMan荧光探针，以重组质粒pMD18-T-IFN-γ和pMD18T-CyPA为标准品，进行实时荧光定量RT-PCR检测，并构建猪IFN-γ mRNA和CyPA的荧光定量RT—PCR标准曲线。结果表明，建立的方法在1×10^1～1×10^7copies／μL模板范围内具有良好的线性关系，相关系数产均高达0．999，扩增效率均高于99．0％；可检测至少为100 copies的阳性标准品。该方法具有快速、敏感性高、特异性强和重复性好等特点，可应用于临床样品的检测。To establish a fluorescent quantitative RT PCR assay for detection of porcine interferon-γ （IFN-γ）, specific primers and fluorescent probes based on porcine IFN-γ gene and housekeeping gene eyelophilin A（CyPA） were designed,respectively. Using the plasmids pMD18-T-IFN-γ and pMD18-T-CyPA as standard products, a real-time quantitative TaqMan reverse transcription-polymerase chain reaction （RTPCR） was performed to construct the standard curves of porcine IFN-γ and CyPA. The results indicated that the assay for the quantitation of porcine IFN-γ template worked well from 15×10^1 to 1 × 10^7 copies/μL with excellent linearity,the coefficient correlation r^2 reached 0. 999 and amplification efficiency was higher than 99.0%. The sensitivity was sufficient to detect the least template of 100 copies. The established porcine IFN-γ specific real-time RT-PCR was time-saving, highly sensitive, specific, reliably reproducible and suitable to detect clinical specimens.

关 键 词：猪 IFN-Γ 荧光定量RT-PCR TaqMan荧光探针 

分 类 号：Q511[生物学—生物化学] Q503