MAS-PCR法快速诊断分析Leber’s遗传性视神经病变原发性致病突变位点  

Rapid genetic analysis in the diagnosis of primary Leber’s hereditary optic neuropathy by using multiplex allele-specific polymerase chain reaction (MAS-PCR) with whole blood

作　　者：阳菊华[1] 童绎[2] 朱益华[2] 林宇岚[1] 陈贻锴[1] 林建银[3] 

机构地区：[1]福建医科大学医药生物工程中心,福建福州350004 [2]福建医科大学第一附属医院眼科,福建福州350004 [3]福建医科大学分子医学研究中心,福建福州350004

出　　处：《眼视光学杂志》2006年第6期349-351,355,共4页Chinese Journal of Optometry & Ophthalmology

基　　金：福建省青年科技人才创新基金资助项目(2004J044)

摘　　要：目的探索一种简易、快速、高效的临床基因诊断分析原发性Leber’s遗传性视神经病变(Leber’s hereditaryoptic neuropathy,LHON)位点的新方法。方法根据已知的LHON患者线粒体DNA上的3个原发性LHON致病突变位点(G11778A、T14484C和G3460A),设计3条突变位点特异性聚合酶链反应(polymerase chain reaction,PCR)引物,直接以全血样为模板的等位基因特异性多重PCR(multiplex allele-specific polymerase chain reaction,MAS-PCR)分析3个原发性LHON致病突变位点。以此方法检测24例确诊的LHON患者,其中18例为线粒体DNA G11778A突变,4例为T14484C突变,2例为G3460A突变;同时,以20份正常血样标本作阴性对照。结果优化的MAS-PCR条件为94℃/3min,94℃/30s、59.8℃/30s、72℃/30s,30个循环,72℃/3min;以此方法检测了44份血样标本,其结果与预期结果一致,表明利用该方法筛查3个原发性LHON致病突变位点是可行的。同时,混和血液样本模拟实验结果显示,该方法可检测含多个原发性LHON致病突变位点的血液样本,且整个分析过程只需约2h。结论该方法直接以全血样作为PCR的模板,不需从血样中纯化DNA;单管一次性行PCR,可同时筛查3个原发性LHON致病突变位点。因此,该方法具有快速、高效、费用低、特异性好以及只需微量血液样本等优点。Objective To establish an effective and rapid generic screen- ing method for diagnosis of Leber＇s hereditary optic neuropathy （IHON） with primary LHON mutation on mitoehondria DNA （mtDNA）. Methods Three allele-specific primers for mtDNA mutations, G11778A, T14484C and G3460A, were designed for multiplex allele-specific polymexase chain reaction （MAS-PCR） with whole blood as a template. Twenty-four blood samples were obtained from ILION patients, who had been diagnosed with ILION associated with one of the three primary mutations in mtDNA （ 18/24 of G11778A, 4/24 of T14484C and 2/24 of C3460A）. In edditian, 20 blood samples free of any eye disorder were obtained as negative controls. Whole blood with anticoagulants was directly used as a template of MAS-PCR for the genetic analysis of IMON. The amplified DNA fragment was directly observed by electrophoretogram with ethidium bromide stain. Results The following procedure was used to optimize the MAS-PCR condition： preheating at 94℃/ 3 min, 30 cycl of 94℃/30 see, 59.8℃/30 see, and 72℃/30 sec, and then a final cycle of 71℃/3 min. The results were completely consistent, showing the presence of the G11778A, T14484C and G3460A mutations in 18,4 and 2 blood samples from LHON patients, respectively, and its absence in 20 normal blood samples by using MAS-PCR with whole blood as a template. This method could test blood saraples containing two or three primary LHON mutations in mtDNA by stimulating blood sample mixtures. And it takes only about two hours to diagnose LHON. Conclusion MAS-PCR with three primers in one. reaction and whole blood as a template is faster and more eest-effective and more convenient as a screening aesay for the dinical genetic diagnosis of LHON.

关 键 词：Leber's遗传性视神经病变 脱氧核糖核酸 线粒体 等位基因特异性多重PCR 基因诊断 

分 类 号：R774.6[医药卫生—眼科]