TaqMan-MGB probe quantitative PCR assays to genotype and quantify three mtDNA mutations of Leber hereditary optic neuropathy

Bingqian Xue,Rui Li,Yi Huang,Yang Li,Liming Bao,Xiaohui Xu,Xin Wang,Tingbo Ye,Xin Zeng,Meihua Yang

doi:10.1038/s41598-020-69220-7

Abstract

Leber hereditary optic neuropathy (LHON) is a degenerative disease of the optic nerve associated with one of three mitochondrial DNA (mtDNA) m.3460G>A, m.11778G>A and m.14484T>C mutations. Although several procedures are available to genotype these mutations, quantitative approaches with rapid, low-cost and easy to handle advantages for three LHON mtDNA mutations are rarely reported. Here, we firstly developed a “one-step” tetra-primer amplification-refractory mutation system (T-ARMS) PCR for qualitative genotyping of three LHON mtDNA mutations. Subsequently, we established single, duplex and triplex TaqMan MGB probe-based fluorescence quantitative PCR (qPCR) assays to perform both qualitative and quantitative analyses of three LHON mtDNA mutations. Standard curves based on tenfold diluted plasmid standard exhibited high specificity and sensitivity, stable repeatability and reliable detectable ability of TaqMan probe qPCR assays without cross-reactivity upon probes combination. Moreover, by comparing with SYBR Green qPCR, we further validated the feasibility of the triplex-probe qPCR assay for the quantitative detection of mtDNA copy number in blood samples. In conclusion, our study describes a rapid, low-cost, easy to-handle, and high-throughput TaqMan-MGB probe qPCR assay to perform both qualitative and quantitative analysis of three primary LHON mtDNA mutations, offering a promising approach for genetic screening and testing of LHON mutations.

Highlights

Leber hereditary optic neuropathy (LHON; MIM 535,000) is a maternally inherited mitochondrial DNA disorder characterized by clinical presentations that include acute or sub-acute vision loss with a male prevalence[1,2]
We developed a “one-step” qualitative PCR technique based on the “Tetra-primer Amplification Refractory Mutation System” (T-ARMS) to define mutation types of three primary LHON mutations, which is suitable for routine pre-screening in conventional laboratories with limited resources
To rapidly genotype three primary LHON mutations, a “one-step” T-ARMS-PCR system was established by a series of optimized programs

Summary

Introduction

Leber hereditary optic neuropathy (LHON; MIM 535,000) is a maternally inherited mitochondrial DNA (mtDNA) disorder characterized by clinical presentations that include acute or sub-acute vision loss with a male prevalence[1,2]. The conventional approaches in genotyping LHON mtDNA mutations are methods that use the PCR product of the DNA fragment containing the mutation, such as denaturing gradient gel electrophoresis(DDGE)[8,9], single-stranded conformational polymorphism (SSCP)[10,11], real-time SYBR Green PCR12,13, temporal temperature gradient gel electrophoresis[14], denaturing high-performance liquid chromatography (DHPLC)[15], solid-phase minisequencing[16], fluorescence probe-based invader assay[17] and PCR-restriction fragment length polymorphism (RFLP)[18] These molecular tools for molecular screening of LHON have become popular, often they are expensive, time-consuming or difficult to handle, and sometimes challenging to reproduce or define precise heteroplasmic levels of LHON mutations. We developed a “one-step” qualitative PCR technique based on the “Tetra-primer Amplification Refractory Mutation System” (T-ARMS) to define mutation types of three primary LHON mutations, which is suitable for routine pre-screening in conventional laboratories with limited resources Following this approach, we established single, duplex and triplex TaqMan-minor groove binder (MGB) probe-based qPCR assays to perform both qualitative and quantitative analysis. Our study describes a rapid, low cost, easy to handle and reliable assay for qualitative and quantitative analysis of three primary LHON mutations

Methods

Results

Conclusion