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[Detection of mtDNA*LHON G11778A mutation by real-time polymerase chain reaction using TaqMan-MGB probe technology]
Yu-min Li1, Jian-yong Wang, Yu Chen
1Zheyi Eye Center, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China. zyec@mail.hz.zj.cn
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.
