摘要
根据猪程序性死亡因子PD-1及其配体PD-L1和PD-L2的基因序列,分别设计了特异性引物和TaqMan探针,以10倍系列稀释的重组质粒pMD-PD-1、pMD-PD-L1和pMD-PD-L2为标准品,对实时荧光定量PCR的条件进行优化,以建立定量检测这3个基因的方法。结果表明,建立的方法在1×10^2-1×10^8copies/μL模板范围内具有良好的线性关系（r^2〉0.99）,扩增效率均高于96%,可检测至少100 copies的阳性标准品。利用该方法对猪外周血单核细胞中这3个基因的表达情况进行了检测,结果也表明该方法具有良好的敏感性、特异性和重复性,可应用于临床样品的检测。
To establish a real-time fluorescent quantitative RT-PCR assay targating on porcine PD-1,PD-L1 and PD-L2 genes,specific primers and fluorescent probes were designed accrodingly.The reaction conditions for the real-time fluorescent quantitative RT-PCR were optimized based on the serially-diluted recombinant plasmids pMD-PD-1,pMD-PD-L1 and pMD-PD-L2.The assay worked well with a good coefficient correlation（r^2〉0.99） and amplification efficiency（〉96%） while the template concentration was from 1×10^2 to 1×10^8 copies/μL.The sensitivity was sufficient to detect at least 100 copies of the samples.The established assay was able to detect the expression of the three genes in porcine peripheral blood mononuclear cell,indicating that the assay was highly sensitive,specific and well reproducible,and suitable for detection of clinical samples.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第4期410-414,共5页
Chinese Veterinary Science
基金
国家重点基础研究发展计划(973)项目(2005CB523202)
兽医生物技术国家重点实验室自主研究项目(SKLVBP201011)
