CAJ | 학술논문

目的建立一种快速检测α地中海贫血(α地贫)SEA缺失的双重TaqMan实时荧光嵌套PCR方法.方法收集2010年5-7月在广州市天河区妇幼保健院进行地贫筛查的外周血标本100份,2010年12月至2011年2月东莞东华医院进行产前诊断的胎儿标本7份(绒毛2份、羊水5份).从本实验室样本资源库中选取α地贫SEA缺失已知基因型标本50份.采用双重TaqMan实时荧光嵌套PCR技术,于同一检测体系同时检测各标本d地贫SEA缺失截短序列及缺失范围内正常序列,根据荧光PCR阳性扩增结果结合其Ct值差异诊断受检个体的α地贫SEA缺失基因型.同时采用检测α地贫SEA缺失的常规gap-PCR法,以PCR扩增结合产物凝胶电泳分析各标本α地贫SEA缺失基因型,以验证及对比分析新方法的准确性与实用性.结果所建立的双重TaqMan实时荧光嵌套PCR优化体系中2个PCR的扩增效率分析曲线的斜率分别为-3.153(SEA缺失截短目的片段)和-3.182(正常内参序列),扩增效率均接近100%.应用此方法检测50份α地贫SEA缺失已知基因型标本,结果显示该方法不但能实现其快速分子诊断,而且能准确判断受检标本中的外源性污染,从而有效避免假阴性或假阳性误诊.100份外周血标本中,两种方法分别检出SEA缺失标本11份,对比分析两方法的诊断符合率为100%.7份胎儿标本中,gap-PCR法检出SEA缺失3份,而本方法则检出SEA缺失标本2份、受-SEA/αα母源性污染的基因型为αα/αα的绒毛标本1份.结论本研究建立的双重TaqMan实时荧光嵌套PCR可以快速准确检测α地贫SEA缺失,操作简单实用,适合大规模人群筛查和常规分子诊断.
목적 건립일충쾌속검측α지중해빈혈(α지빈)SEA결실적쌍중TaqMan실시형광감투PCR방법.방법 수집2010년5-7월재엄주시천하구부유보건원진행지빈사사적외주혈표본100빈,2010년12월지2011년2월동완동화의원진행산전진단적태인표본7빈(융모2빈、양수5빈).종본실험실양본자원고중선취α지빈SEA결실이지기인형표본50빈.채용쌍중TaqMan실시형광감투PCR기술,우동일검측체계동시검측각표본d지빈SEA결실절단서렬급결실범위내정상서렬,근거형광PCR양성확증결과결합기Ct치차이진단수검개체적α지빈SEA결실기인형.동시채용검측α지빈SEA결실적상규gap-PCR법,이PCR확증결합산물응효전영분석각표본α지빈SEA결실기인형,이험증급대비분석신방법적준학성여실용성.결과 소건립적쌍중TaqMan실시형광감투PCR우화체계중2개PCR적확증효솔분석곡선적사솔분별위-3.153(SEA결실절단목적 편단)화-3.182(정상내삼서렬),확증효솔균접근100%.응용차방법검측50빈α지빈SEA결실이지기인형표본,결과현시해방법불단능실현기쾌속분자진단,이차능준학판단수검표본중적외원성오염,종이유효피면가음성혹가양성오진.100빈외주혈표본중,량충방법분별검출SEA결실표본11빈,대비분석량방법적진단부합솔위100%.7빈태인표본중,gap-PCR법검출SEA결실3빈,이본방법칙검출SEA결실표본2빈、수-SEA/αα모원성오염적기인형위αα/αα적융모표본1빈.결론 본연구건립적쌍중TaqMan실시형광감투PCR가이쾌속준학검측α지빈SEA결실,조작간단실용,괄합대규모인군사사화상규분자진단.
Objective To establish a double TaqMan real-time fluorescence nested PCR method for rapid detection of α-thalassemia SEA deletion.Methods One hundred blood samples for thalassemia screening were collected from May to July of 2010 in the Tianhe Maternal and Child Health Hospital of Guangzhou.Seven fetal specimens for prenatal diagnosis were collected from December 2010 to February 2011 in Dongguan TungWah Hospital(2 villi and 5 amniotic fluid specimens).Fifty samples of α-thalassemia SEA deletion with genotyping results were selected from the sample bank of our laboratory.The double TaqMan real-time fluorescence nested PCR was applied to detect the truncated sequence of SEA deletion and the normal sequence within deletion range simultaneously for all these samples with the same detecting system.The genotype of α-thalassemia SEA deletion was accurately acquired according to the positive result of fluorescent PCR combined with the Ct value difference.Meanwhile,the accuracy and feasibility of this method were verified and analyzed by parallely detecting these samples with routine gapPCR for α-thalassemia SEA deletion.The genotype could be obtained according to PCR amplification and agarose gel electrophoresis.Results Two amplification efficiencies of the optimized dual TaqMan real-time fluorescence nested PCR system established in this study were both close to 100% with the slops of -3.153 and -3.182,respectively.The results of 50 samples of α-thalassemia SEA deletion with genotyping results showed that this method could not only realize rapid diagnosis,but also effectively avoid false negative or false-positive misdiagnosis by accurately determine the external contamination in the sample.Among 100 blood samples,eleven samples with SEA deletion were detected respectively and the diagnosis coincidence rate of these two methods was 100%,3 samples with SEA deletion were detected by gap-PCR,but 2 samples with SEA deletion and 1 villi sample with normal genotype but contaminated by SEA were detected by this method among 7 fetal samples.Conclusions A double TaqMan real-time fluorescence nested PCR method for α-thalassemia SEA deletion was developed.The method is a rapid and reliable test with simple operation,and is suitable for large-scale population screening and routine molecular diagnosis.