建立RT-ARMS-qPCR系统定量测定含A1555G突变的线粒体DNA拷贝数

目的建立RT-ARMS-qPCR（real time-amplification refractory mutation system-quantitative PCR）系统定量测定含线粒体DNA（mitochondrial DNA,mtDNA）A1555G位点突变的片段的拷贝数,为阐明线粒体耳聋临床表型多样性的分子生物学基础奠定基础。方法根据ARMS的基本原理设计引物,在引物3’端插入错配碱基AC建立RT-ARMS-qPCR系统,优化定量PCR体系,并进行方法学评价。经PCR分别扩增相应突变型和野生型的片段,将其克隆到pGEM-T Easy载体上,构建质粒标准品;同时对含突变型和/或野生型mtDNA 1555位点的片段的拷贝数进行定量检测。结果RT-ARMS-qPCR系统用于检测含mtDNA 1555位点的片段,线性检测范围为102-108拷贝数/μl,检测灵敏度为1×102拷贝数/μl,其批内变异系数（CV）为1.34%,批间CV为1.96%,重复性好;突变型和野生型引物分别只扩增相应片段,特异性好。结论RT-ARMS-qPCR系统适合于定量检测含mtDNA A1555G点突变的线粒体DNA片段,结果稳定、准确,有助于阐明线粒体耳聋严重程度的分子基础。

Quantitation of mitochondrial DNA copies with mutated 1555 site by real time- Amplification Refractory Mutation System-quantitative PCR

Objective To develop a real-time quantitative PCR method with SYBR Green I to assess the mtDNA 1555.Methods A specific fragment mtDNA 1555 was amplified with PCR and ligated into a pGEM Easy T vector.Real time-Amplification refractory mutation system-quantitative PCR（RT-ARMS-qPCR） was established to detect the number of mtDNA copies containing mild type and mutant type mtDNA 1555. The serial dilutions of the plasmid DNAs were quantified the actual copy numbers using Real time-Amplification refractory mutation system-quantitative PCR. The specificity of amplified products was checked by melting curve analysis. Results From 1 ×10^8 copies/μl to 10 copies/μl of 10^2 fold serial diluted plasmid DNA could be quantified with real-time quantitative PCR. The quantitative standard curve showed that they had good linear correlation and could be served for the quantification of other samples. Conclusion The results of mtDNA 1555 copy number detected by real time-Amplification Refractory Mutation System-quan- titative PCR are stable and accurate.