摘要目的 针对线粒体糖尿病致病位点mtDNA 3243A→G异质性突变,建立一种快速简便、低成本,但相对准确、敏感的定量检测方法,为不同条件下选择最佳检测方案提供参考.方法 收集浙江省温州市一个母系遗传性线粒体糖尿病伴耳聋家系(maternally inherited diabetes and deafness MIDD)共17人,提取外周血全基因组DNA,同时分别应用PCR-限制性片段长度多态性技术(polymerase chain reactionrestriction fragment length polymorphism PCR-RLFP)、实时荧光定量PCR结合突变阻滞形成系统(real time-amplification refractory mutation system-quantitative PCR,RT-ARMS-qPCR)和焦磷酸测序技术检测mtDNA A3243G突变的异质水平;并以0～100％不同比例A3243G突变负荷的11个标准品为对照,3种方法同时分别检测,根据实际检测值与预期检测值的相关程度,对这三种检测方法做出可靠性比较分析.结果 PCR-RFLP、RT ARMS-qPCR和焦磷酸测序技术对A3243G定量标准品的检测结果中,PCR-RFLP相对定量分析结果与预期值之间呈直线相关,决定系数R2=0.828;RT-ARMS-qPCR检测11个标准对照的突变负荷结果与预期值之间呈直线相关,决定系数R2 =0.998;焦磷酸测序技术检测结果与预期值之间呈直线相关,决定系数R2=0.997.定量检测A3243G异质水平结果PCR-RFLP介于10％～60％,RTARMS-qPCR为0到100％,焦磷酸测序技术为1％到95％;RT ARMS-qPCR和焦磷酸测序技术的检测结果均接近预期值;针对MIDD家系17位成员的检测中A3243G突变携带者有13例,非A3243G突变携带者有4份,这三种方法定性检测结果一致.RT-ARMS-qPCR和焦磷酸测序技术两种方法定量检测结果差异无统计学意义(t=1.140,P＞0.05).结论 针对mtDNA 3243A→G异质性突变的定量检测方法,PCR-RFLP不适合定量检测,但可作为一种临床检测方法用于人群初步筛查.对于低异质水平的A3243G突变可用RT ARMS-qPCR和焦磷酸测序技术两种方法检测,但RT ARMS-qPCR相对于焦磷酸测序技术更简便、快速、可靠,成本低,是普通实验室和临床检测低异质负荷的最佳选择方案.

abstractsObjective To develop a rapid,simple,cost-effective,accurate and sensitive method for quantitative detection of mitochondrial DNA (mtDNA) 3243A→G mutation in order to provide reference for selecting the best detection method under different conditions.Methods Genomic DNA was extracted from peripheral leucocytes of 17 individuals from a Wenzhou family featuring maternally inherited diabetes and deafness (MIDD).Heteroplasmic level of mtDNA 3243A→G mutation was determined respectively with polymerase chain reaction-restriction fragment length polymorphism (PCR-RLFP),real time-amplification refractory mutation system-quantitative PCR (RT-ARMS-qPCR) and pyrosquencing.Eleven plasmids with various heteroplasmic levels of the 3243A→G mutation (ranging from 0 to 100％) were constructed as the standards.The reliability of above methods was compared by correlation coefficient based on observed and expected values.Results For all three methods,measurement of the standards showed a linear correlation between the expected and detected values,i.e.,PCR-RFLP (R2 =0.828),RT-ARMS-qPCR (R2 =0.998) and pyrosquencing (R2 =0.997).For the MIDD family,it was consistent that there are 13 members carrying the A3243G mutation with different heteroplasmic levels.And there was no significant difference between the results by RT-ARMS-qPCR and pyrosquencing.Conclusion PCR-RFLP is not appropriate for the quantitative detection but could be used for early clinical screening.Both RT-ARMS-qPCR and pyrosquencing are suitable for the detection of low heteroplasmic level of A3243G mutation.Compared with pyrosquencing,RT-ARMS-qPCR is rapid,reliable and cost-effective,and is the best choice for detecting low mutation loads.