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目的建立一套快速、准确、简便筛查Leber遗传性视神经病变(LHON)mtDNA C11778A基因突变的新方法,并初步判断其异质性个体中野生和突变比例。方法采用荧光MGB探针实时聚合酶链反应(PCR)技术,在同一个反应体系中引入野生和突变型两种探针,应用多荧光通道PCR仪分别检测LHON家系中20例患者血样和17例正常人血样,将检测结果与核酸序列测定结果比较。结果正常人血样检测结果:荧光PCR技术检测VIC通道均为阳性,FAM通道均为阴性,判断为LHON mtDNA野生型,与测序结果完全吻合;临床LHON患者及家系成员测序结果:LHON mtDNA变异型患者12例,荧光PCR技术检测均为LHON mtDNA变异阳性,其中为LHON mtDNA变异株和野生株混合的5例,混合型中变异株与野生株Ct值均大于25％;测序结果显示LHON mtDNA野生型8例,荧光PCR技术检测LHON mtDNA野生型为5例,LHON mtDNA变异株和野生株混合型3例,其中变异株与野生株Ct值均小于25％;荧光定量PCR技术检测LHON mtDNA G11778A基因突变耗时仅为80 min,远短于测序时间。结论荧光MGB探针实时PCR技术能简便、快速、准确地检测LHON mtDNA C11778A基因突变,判断个体异质性中野生和突变比例,对进一步探讨LHON患者是否存在mtDNA基因突变阈值有重要价值。
OBJECTIVE: To establish a rapid, accurate and simple method for screening mutations in the mtDNA C11778A gene of Leber’s hereditary optic neuropathy (LHON) and to determine the proportion of wild-type and mutant individuals in heterogeneous individuals. Methods Real-time fluorescence polymerase chain reaction (MGB) was used to detect wild-type and mutant-type probes in the same reaction system. Twenty blood samples from LHON pedigrees and 17 Normal blood samples, the test results and nucleic acid sequence determination results. Results The blood samples of normal subjects were detected by Fluorescent PCR and all the FAM channels were negative. The LHON mtDNA wild type was judged to be completely consistent with the sequencing results. Sequencing results of LHON patients and their families: LHON mtDNA mutation patients 12 cases were detected by fluorescent PCR technique LHON mtDNA mutation was positive, which LHON mtDNA mutant and wild strains mixed in 5 cases, the mixed mutant Ct values ?? and wild strains were greater than 25%; sequencing results LHON mtDNA wild type 8 cases. LHON mtDNA wild type was detected by fluorescence PCR in 5 cases, LHON mtDNA mutant and wild type mixed in 3 cases, of which the Ct values of the mutant and wild strains were less than 25%; Fluorescent quantitative PCR was used to detect LHON mtDNA G11778A gene mutation Time-consuming only 80 min, much shorter than the sequencing time. Conclusion Real-time PCR with fluorescent MGB probe can detect the mutation of LHON mtDNA C11778A gene easily and rapidly and determine the ratio of wild-type and mutation in individual heterogeneity. It is of great value to further explore the threshold of mtDNA mutation in LHON patients.