中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
CHINESE JOURNAL OF MEDICAL GENETICS
2014年
4期
438-443
,共6页
张小勤%陈琳%杨宇%郑超%颜景斌%路兆宁%吕建新%李伟
張小勤%陳琳%楊宇%鄭超%顏景斌%路兆寧%呂建新%李偉
장소근%진림%양우%정초%안경빈%로조저%려건신%리위
线粒体DNA%PCR-限制性片段长度多态性技术%实时荧光定量PCR结合突变阻滞形成系统%焦磷酸测序技术
線粒體DNA%PCR-限製性片段長度多態性技術%實時熒光定量PCR結閤突變阻滯形成繫統%焦燐痠測序技術
선립체DNA%PCR-한제성편단장도다태성기술%실시형광정량PCR결합돌변조체형성계통%초린산측서기술
Mitochondrial DNA%Polymerase chain reaction-restriction fragment length polymorphism%Real time-amplification refractory mutation system-quantitative PCR%Pyrosquencing
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.
目的 針對線粒體糖尿病緻病位點mtDNA 3243A→G異質性突變,建立一種快速簡便、低成本,但相對準確、敏感的定量檢測方法,為不同條件下選擇最佳檢測方案提供參攷.方法 收集浙江省溫州市一箇母繫遺傳性線粒體糖尿病伴耳聾傢繫(maternally inherited diabetes and deafness MIDD)共17人,提取外週血全基因組DNA,同時分彆應用PCR-限製性片段長度多態性技術(polymerase chain reactionrestriction fragment length polymorphism PCR-RLFP)、實時熒光定量PCR結閤突變阻滯形成繫統(real time-amplification refractory mutation system-quantitative PCR,RT-ARMS-qPCR)和焦燐痠測序技術檢測mtDNA A3243G突變的異質水平;併以0～100％不同比例A3243G突變負荷的11箇標準品為對照,3種方法同時分彆檢測,根據實際檢測值與預期檢測值的相關程度,對這三種檢測方法做齣可靠性比較分析.結果 PCR-RFLP、RT ARMS-qPCR和焦燐痠測序技術對A3243G定量標準品的檢測結果中,PCR-RFLP相對定量分析結果與預期值之間呈直線相關,決定繫數R2=0.828;RT-ARMS-qPCR檢測11箇標準對照的突變負荷結果與預期值之間呈直線相關,決定繫數R2 =0.998;焦燐痠測序技術檢測結果與預期值之間呈直線相關,決定繫數R2=0.997.定量檢測A3243G異質水平結果PCR-RFLP介于10％～60％,RTARMS-qPCR為0到100％,焦燐痠測序技術為1％到95％;RT ARMS-qPCR和焦燐痠測序技術的檢測結果均接近預期值;針對MIDD傢繫17位成員的檢測中A3243G突變攜帶者有13例,非A3243G突變攜帶者有4份,這三種方法定性檢測結果一緻.RT-ARMS-qPCR和焦燐痠測序技術兩種方法定量檢測結果差異無統計學意義(t=1.140,P＞0.05).結論 針對mtDNA 3243A→G異質性突變的定量檢測方法,PCR-RFLP不適閤定量檢測,但可作為一種臨床檢測方法用于人群初步篩查.對于低異質水平的A3243G突變可用RT ARMS-qPCR和焦燐痠測序技術兩種方法檢測,但RT ARMS-qPCR相對于焦燐痠測序技術更簡便、快速、可靠,成本低,是普通實驗室和臨床檢測低異質負荷的最佳選擇方案.
목적 침대선립체당뇨병치병위점mtDNA 3243A→G이질성돌변,건립일충쾌속간편、저성본,단상대준학、민감적정량검측방법,위불동조건하선택최가검측방안제공삼고.방법 수집절강성온주시일개모계유전성선립체당뇨병반이롱가계(maternally inherited diabetes and deafness MIDD)공17인,제취외주혈전기인조DNA,동시분별응용PCR-한제성편단장도다태성기술(polymerase chain reactionrestriction fragment length polymorphism PCR-RLFP)、실시형광정량PCR결합돌변조체형성계통(real time-amplification refractory mutation system-quantitative PCR,RT-ARMS-qPCR)화초린산측서기술검측mtDNA A3243G돌변적이질수평;병이0～100％불동비례A3243G돌변부하적11개표준품위대조,3충방법동시분별검측,근거실제검측치여예기검측치적상관정도,대저삼충검측방법주출가고성비교분석.결과 PCR-RFLP、RT ARMS-qPCR화초린산측서기술대A3243G정량표준품적검측결과중,PCR-RFLP상대정량분석결과여예기치지간정직선상관,결정계수R2=0.828;RT-ARMS-qPCR검측11개표준대조적돌변부하결과여예기치지간정직선상관,결정계수R2 =0.998;초린산측서기술검측결과여예기치지간정직선상관,결정계수R2=0.997.정량검측A3243G이질수평결과PCR-RFLP개우10％～60％,RTARMS-qPCR위0도100％,초린산측서기술위1％도95％;RT ARMS-qPCR화초린산측서기술적검측결과균접근예기치;침대MIDD가계17위성원적검측중A3243G돌변휴대자유13례,비A3243G돌변휴대자유4빈,저삼충방법정성검측결과일치.RT-ARMS-qPCR화초린산측서기술량충방법정량검측결과차이무통계학의의(t=1.140,P＞0.05).결론 침대mtDNA 3243A→G이질성돌변적정량검측방법,PCR-RFLP불괄합정량검측,단가작위일충림상검측방법용우인군초보사사.대우저이질수평적A3243G돌변가용RT ARMS-qPCR화초린산측서기술량충방법검측,단RT ARMS-qPCR상대우초린산측서기술경간편、쾌속、가고,성본저,시보통실험실화림상검측저이질부하적최가선택방안.
Objective 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.
