荧光探针法检测Leber遗传性视神经病变11778位点突变的研究  

Study on real-time probe technique detecting mtDNA 11778 point mutation in Leber hereditary optic neuropathy patients

作　　者：张艳敏[1] 宋丹[1] 安慧娟[1] 陈慷[1] 王志立[1] 鲍玉洲[1] 赵朝霞[1] 

机构地区：[1]河南省眼科研究所河南省立眼科医院(张艳敏,现在河南省中医院检验科),郑州450003

出　　处：《中华实验眼科杂志》2013年第2期164-167,共4页Chinese Journal Of Experimental Ophthalmology

基　　金：河南省重点科技攻关计划项目（112102310004）

摘　　要：背景Leber遗传性视神经病变（LHON）与视神经炎的病因和治疗方法均不相同，但由于其临床表现相似，因此易被误诊。利用实时荧光定量PCR技术建立一种高通量、简便、快速、特异性高的LHON检测方法具有重要的临床意义。目的建立以实时荧光定量突变特异性Taqman探针技术检测LHON线粒体DNA（mtDNA）11778G〉A突变的方法，实现快速、简便、准确地筛查mtDNA11778位点突变LHON患者。方法根据mtDNA全基因组序列设计针对mtDNA11778G〉A突变的特异性Taqman探针和引物，从河南省眼科研究所分子生物室LHONDNA库任意选取标本84份，另抽取40名18～20岁健康体检者的血样40份作为正常对照，分别用实时荧光定量特异性探针法和序列测定法检测LHONmtDNA11778G〉A突变，并对检测方法进行可靠性验证。结果用实时荧光定量Taqman探针法检测mtDNA11778G〉A突变，84例视神经病变患者中发现mtDNA11778G〉A突变者23例，阴性结果者61例，23例阳性患者样本扩增曲线结果显示ct值为22．993±0．708，正常对照组样本ct值均为0，与直接测序法检测结果完全相符，其符合率为100％，未发现假阳性和假阴性结果者。结论用实时荧光定量突变特异性Taqman探针法检测LHONmtDNA11778G〉A突变位点的方法简单、省时、准确、直观、特异性强、敏感度高，适用于对LHONmtDNA11778G〉A突变的大规模筛查或预防性检查。Background Although Leber hereditary optic neuropathy （LHON） and optic neuritis have different causes and managements, their clinical manifestations are difficult to be distinguished. Real-time fluorescence quantitative polymerase chain reaction （RTFQ-PCR） is a high flux, simple, rapid and specific detecting technology, so establishing a specific diagnosis method of LHON with RTFQ-PCR has a practical significance. Objective Purpose of the present study was to establish a real-time Taqman probe for mitochondrial DNA （mtDNA）11778G〉A mutation in LHON patients. Methods Primers and Taqman probe for mtDNA l1778G〉A mutation were designed based on mtDNA complete genemc. Eighty-four patients with LHON were selected from the LHON DNA bank of Molecular Biology Laboratory,Henan Eye Institute, and 40 normal physical examinees aged 18-20 years were from Henan People＇s Hospital. 2 ml of periphery blood was collected from each individual. Based on the double-blindness principle,mtDNA l1778G〉A mutation was tested by both Taqman probe and sequencing to check the reliability of real-time Taqman probe. Results The mtDNA l1778G〉A mutation was found in 23 out of 84 patients,and 61 showed a negative result by the technique of real-time Taqman probe. The Ct values of 23 patients with mtDNA l1778G〉A mutation were 22. 993＋0. 708 ,but those of 5 normal controls were 0. These findings showed a consistent rate of 100% with the sequencing results. In addition,both the false positive rate and the false negative rate were zero. Conclusions Real-time Taqman probe technique is an method fi）r the diagnosis of mtDNA l1778G〉A mutation in LHON patients with mtDNA I1778G〉A mutation in a large aecurate, convenient, sensitive, specific and intuitionistic LHON patients. It is feasible and suitable to screen the scale.

关 键 词：LEBER遗传性视神经病变 线粒体DNA 实时荧光定量PCR 点突变 

分 类 号：R774.6[医药卫生—眼科]