实时定量PCR检测KIR转录水平的方法构建  

Method construction of quantitative real-time PCR for detecting KIR mRNA levels

作　　者：陈鸥[1,2] 陈会会[1] 姜拥军[1] 尚红[1] 

机构地区：[1]中国医科大学附属第一医院检验科艾滋病研究所,辽宁沈阳110001 [2]中国医科大学附属第四医院检验科

出　　处：《中国实验诊断学》2015年第12期2030-2033,共4页Chinese Journal of Laboratory Diagnosis

基　　金：国家科技重大专项课题资助(2008ZX10001-001)

摘　　要：目的构建可定量检测杀伤细胞免疫球蛋白样受体(KIR)转录水平的方法。方法筛选KIR3DS1和KIR3DL1保守区特异性引物,利用RT-PCR和熔解曲线法对引物特异性进行检验,建立可定量的标准内参,应用SYBR Green RT-PCR方法检测标本中KIR3DS1和KIR3DL1的mRNA含量。结果通过电泳和实时定量PCR的熔解曲线明确了KIR3DS1和KIR3DL1引物,其特异性好,无杂带。应用TA克隆建立的KIR定量标准品线性关系好,可检测待测物的范围大。应用临床标本可检测出两组标本有显著差异,符合临床预期。结论本文建立的KIR mRNA定量检测方法,可以定量检测活化和抑制性KIR,可以进一步研究不同疾病进展的机制以及预测疾病的临床转归。To build quantitative real-time PCR for detecting（Killer cell Ig-like receptors,KIR）mRNA levels with SYBR Green method,we screened primers of KIR3DS1 and KIR3DL1that aimed at the specific conservative region.Our experiment used PCR,electrophoresis and melting curve to find out the specificity of the primers and set up quantitative standard reference.Then we applied TA clone to build KIR quantitative standard reference which had a good linear relationship and a large detection range.Patients＇ samples were tested with statistical differences between two groups and the results were in accordance with clinical expectation.In conclsion：the method we built can quantitatively detect both active and inhibitive mRNA levels of KIR and can people further understand the progress and clinical outcome of diseases.

关 键 词：KIR基因 KIR3DS1 KIR3DL1 实时定量PCR 

分 类 号：R440[医药卫生—诊断学] R512.91[医药卫生—临床医学]