Establishment And Evaluation Of Quantitative Analysis Of LHON Mutant MtDNA Copy Number

Objective: Leber hereditary optic neuropathy(LHON)is an optic nerve degenerative disease.In the Chinese Han population,it is mainly associated with three mitochondrial DNA(mtDNA)mutations: m.3460G> A,m.11778G> A,and m.14484T> C.Although there are several methods for genotyping these mutations,such as: SSCP-PCR,RFLP-PCR,and DNA sequencing,etc.,due to expensive,complicated procedures,and time-consuming factors,their use in clinical applications has been limited.Few quantitative methods have been reported for three LHON mtDNA mutations with the advantages of fast,low cost,and easy handling.Therefore,in this study,a TaqMan small groove conjugate(MGB)probe-based fluorescent quantitative PCR(qPCR)method was developed to perform qualitative and quantitative analysis of three LHON mtDNA mutations.Methods: 1.We construct positive control standards and negative control standards for the three main mutation types of mtDNA.Primers and probes were designed according to the design principles of TaqMan-MGB probe qPCR primer probes.2.We established an amplification system that can detect single/ duplex /triplex probes in a single tube by optimizing the annealing temperature and the ratio of primers and probes.Then we tested the specificity,sensitivity,detectable ratio of each amplification system and created a standard curve.3.We collected 3 positive cases of LHON mutation(1 for m.3460G>A,1 for m.11778G>A,and 1 for m.14484T>C)and 45 negative cases.TaqMan-MGB probe qPCR was used to verify their genotypes.The positive control standard was mixed with the normal genomic DNA sample to make a mock sample with a mutation content of 5% to 95%.The TaqMan-MGB probe qPCR was used for quantitative detection to evaluate its ability to detect mutation heterogeneity.Results: 1.The positive standards and negative standards for three types of mtDNA mutation were successfully constructed,and TaqMan-MGB probe qPCR probes and primers were designed.2.The TaqMan-MGB probe qPCR system was successfully established.3.Three positive cases of LHON mutation(1 for m.3460G>A,1 for m.11778G>A,and 1 for m.14484T>C)and 45 negative cases were successfully detected.Simulation samples with 5% ~ 95% mutation content were also successfully detected.Conclusion: Based on the TaqMan-MGB probe qPCR,we have successfully established a new LHON detecting method with good specificity,sensitivity,detection rate,and clinical application performance.This method has.This method also has the advantages of fast,low cost and easy handling.