荧光MGB探针在Leber遗传性视神经病变mtDNA G11778A基因突变检测中的应用

Detection of mtDNA LHON G11778A mutation by real-time polymerase chain reaction using TaqMan-MGB probe technology

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 develop a simple, rapid and reliable real-time PCR assay based on TaqMan technology using a new MGB probe for detecting mtDNA^＊ LHON G11778A mutation and heteroplasmy directly. Methods Twenty patients with suspicion of Leber hereditary optic neuropathy （LHON） and their maternal relatives had undergone molecular genetic evaluation. Seventeen normal individuals were used as the controls. A real-time PCR involved two MGB probes （wild-type and mutation- type） in a single tube on the iCycler IQ real-time detection system was used to detect the mtDNA^＊ LHON G11778A mutation. The results were then compared with the DNA sequence analysis of the PCR products. A linear standard curve was obtained by pUCm LHON-G and pUCm LHON-A clone. Results In the controls （wild type） , the reaction of VIC-labeled MGB probe was positive and the channel of FAM reaction was negative, the DNA sequence was 100% matched to previously published data. In 20 LHON patients and their maternal relatives, 12 cases showed mutations in DNA sequence analysis, all of them were LHON mtDNA mutation. While 5 other cases showed the combination of LHON mtDNA mutation and wide type gene phenotype, the rate of Ct value in wild type versus gene mutation was over 25%. DNA sequence analysis showed 8 of LHON mtDNA belonged to wild types and 3 cases were heteroplasmy, and the rate of Ct value in gene mutation versus wild type was lower than 25%. Conclusion This real-time PCR assay is a simple, rapid and reliable method for the detection of genotyping mtDNA mutations as well as for quantifying heteroplasmy. （Chin J Ophthalmol , 2006,42 ： 728-732 ）