CAJ | 학술논문

目的应用实时荧光定量PCR结合突变阻滞形成系统(RT ARMS-qPCR)技术检测线粒体DNA A3243G突变负荷,并探讨线粒体脑肌病伴高乳酸血症和脑卒中样发作(MELAS)综合征患者不同组织中A3243G突变负荷的分布情况.方法分别构建含线粒体DNA3243位点的野生型(A)和突变型(G)序列的质粒,将这2种质粒按一定比例混合构建成13个含有不同A3243G突变负荷的标准对照,应用PCR-限制性片段多态性技术(RFLP)和RT ARMS-qPCR技术检测13个标准对照及存在A3243G突变的7例MELAS患者、1名携带者以及53名健康对照的外周血、肌肉、毛发及尿沉渣等组织的A3243G突变负荷.结果标准对照的突变负荷经PCR-RFLP和RT ARMS-qPCR检测的结果均与预期值呈直线相关,决定系数分别为R21=0.885、R22=0.991,RT ARMS-qPCR检测的结果更接近预期值;7例MELAS患者及1名携带者不同组织的突变负荷经RT ARMS-qPCR检测的结果比经PCR-RFLP检测出的高,且RT ARMS-qPCR比PCR-RFLP更容易检测出低负荷的A3243G突变,并发现MELAS患者的肌肉、尿沉渣、头发的A3243G突变负荷均高于外周血.结论应用RT ARMS-qPCR可以快速、灵敏、准确地检测不同组织的A3243G突变及其突变负荷.
목적 응용실시형광정량PCR결합돌변조체형성계통(RT ARMS-qPCR)기술검측선립체DNA A3243G돌변부하,병탐토선립체뇌기병반고유산혈증화뇌졸중양발작(MELAS)종합정환자불동조직중A3243G돌변부하적분포정황.방법 분별구건함선립체DNA3243위점적야생형(A)화돌변형(G)서렬적질립,장저2충질립안일정비례혼합구건성13개함유불동A3243G돌변부하적표준대조,응용PCR-한제성편단다태성기술(RFLP)화RT ARMS-qPCR기술검측13개표준대조급존재A3243G돌변적7례MELAS환자、1명휴대자이급53명건강대조적외주혈、기육、모발급뇨침사등조직적A3243G돌변부하.결과 표준대조적돌변부하경PCR-RFLP화RT ARMS-qPCR검측적결과균여예기치정직선상관,결정계수분별위R21=0.885、R22=0.991,RT ARMS-qPCR검측적결과경접근예기치;7례MELAS환자급1명휴대자불동조직적돌변부하경RT ARMS-qPCR검측적결과비경PCR-RFLP검측출적고,차RT ARMS-qPCR비PCR-RFLP경용역검측출저부하적A3243G돌변,병발현MELAS환자적기육、뇨침사、두발적A3243G돌변부하균고우외주혈.결론 응용RT ARMS-qPCR가이쾌속、령민、준학지검측불동조직적A3243G돌변급기돌변부하.
Objective To evaluate the quantitative technique of real-time amplification refractory mutation system quantitative PCR( RT ARMS-qPCR)in the detection of the mitochondrial DNA A3243G mutation load. To investigate the mutation load in different tissues in patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). Methods Wild type and mutant-type (A3243G) of mitochondrial DNA were cloned into plasmid pMD18-T to construct express vectors. Thirteen standard controls having different proportions of mutation loads were developed by mixing wild-type and mutant-type cloned plasmid DNA in different ratios. The mutation loads in the tissues of blood, muscle, hair follicles and urine from seven patients with MELAS and one carrier, and blood samples in 53 unaffected subjects were detected by lit ARMS-qPCR and PCR-RFLP. Results In standard controls, there was a linear correlation between the expected values and results of mutation loads detected by both methods of PCR-RFLP (R21 = 0. 885 ) and RT ARMS-qPCR (R22 = 0. 991 ) . The results detected by RT ARMS-qPCR were closer to the expected values. The detection of mutation loads in tissues from the patients revealed higher values by liT ARMS-qPCR method than by PCR-RFLP and RT ARMS-qPCR was more sensitive in detecting the lower A3243G mutation load. The mutation load in muscle, hair follicles or urinary sedimem is higher than that in leukoeytas. Conclusion The RT ARMS-qPCR provides a convenient,rapid, sensitive and reliable quantitative detection of heteroplasmic mutant mtDNA A3243G in different tissues.