[Detection of mtDNA*LHON G11778A mutation by real-time polymerase chain reaction using TaqMan-MGB probe technology].
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%.


CONCLUSIONS
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.
Li
Yu-min
YM
Zheyi Eye Center, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China. zyec@mail.hz.zj.cn
Wang
Jian-yong
JY
Chen
Yu
Y
Shao
Jun-bin
JB
Wang
Jing
J
Tong
Yi
Y
D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D006801:Humans; D008297:Male; D015341:Nucleic Acid Probes; D029242:Optic Atrophy, Hereditary, Leber / Q000175:diagnosis / Q000235:genetics*; D010375:Pedigree; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase
D004272:DNA, Mitochondrial; D015341:Nucleic Acid Probes; D019914:Taq Polymerase