CAJ | 학술논문

目的建立基因点突变双重TaqMan荧光定量PCR方法，实现β-地中海贫血（β-地贫）高风险胎儿产前基因诊断快速、准确。方法以每反应管检测一个位点的突变和野生型基因序列，建立β-地贫基因点突变双重TaqMan荧光定量PCR体系，检测β-地贫高风险胎儿绒毛标本6例、羊水标本32例，根据荧光PCR阳性扩增结果结合两荧光通道的Ct值差异分析受检标本的基因型，同时以常规RDB检测为对照，分析与评价检测结果。结果6例胎儿绒毛标本中，RDB检测无法判断结果1例，经该方法检测为野合纯合子；RDB检测误诊为CD41-42合并CD26双重杂合子1例，经该方法检测为CD41-42杂合子。32例胎儿羊水标本中， RDB检测无法判断的标本1例，经该方法检测为CD41-42杂合子。该3例标本经重新采集胎儿羊水标本复查，验证了定量检测结果的准确性。结论β基因点突变双重TaqMan荧光PCR定量检测方法，可实现β-地贫高风险胎儿的快速准确产前基因诊断，操作简单快速，结果准确可靠，是较为理想的β-地贫产前诊断方法。
목적건립기인점돌변쌍중TaqMan형광정량PCR방법，실현β-지중해빈혈（β-지빈）고풍험태인산전기인진단쾌속、준학。방법이매반응관검측일개위점적돌변화야생형기인서렬，건립β-지빈기인점돌변쌍중TaqMan형광정량PCR체계，검측β-지빈고풍험태인융모표본6례、양수표본32례，근거형광PCR양성확증결과결합량형광통도적Ct치차이분석수검표본적기인형，동시이상규RDB검측위대조，분석여평개검측결과。결과6례태인융모표본중，RDB검측무법판단결과1례，경해방법검측위야합순합자；RDB검측오진위CD41-42합병CD26쌍중잡합자1례，경해방법검측위CD41-42잡합자。32례태인양수표본중， RDB검측무법판단적표본1례，경해방법검측위CD41-42잡합자。해3례표본경중신채집태인양수표본복사，험증료정량검측결과적준학성。결론β기인점돌변쌍중TaqMan형광PCR정량검측방법，가실현β-지빈고풍험태인적쾌속준학산전기인진단，조작간단쾌속，결과준학가고，시교위이상적β-지빈산전진단방법。
Objective To establish a dual Taqman fluorescence quantitative PCR ( qPCR ) method for point mutation detection , and to realize the fast and accurate prenatal diagnosis of high-risk fetus with beta-thalassemia . Methods A dual Taqman qPCR method for beta-thalassimia point mutation detection was developed according to the design that mutation and wild type sequences of a mutation site analyzed in one reaction tube .This method was also applied to detect 6 villi and 32 amniotic fluid specimens of high-risk fetus with beta-thalassemia .The genotype of a sample was analyzed according to the positive amplification results of qPCR and Ct variance of two fluorescent chan -nels.Meanwhile,reverse dot blot(RDB) method was used as a control for the analysis and evaluation of detection re-sults.Results Of 6 villi specimens ,1 sample which could not be diagnosed with RDB method was detected wild type homozygote by this method ,and another sample with the misdiagnosis RDB result of double heterozygote of βCD41-42 and βCD26 was detected heterozygote of βCD41-42 by this method .Of 32 amniotic fluid specimens ,1 sample which could not be diagnosed with RDB method was detected heterozygote of βCD41-42 by this method .All these 3 samples were also re-analyzed and verified with recollected amniotic fluid specimens .Conclusion The fast and accurate pre-natal diagnosis of high-risk fetus with beta-thalassemia can be realized with this dual Taqman qPCR method for beta gene point mutation developed in this study .The method is accurate with easy operation and suitable for prenatal diag-nosis of beta-thalassemia .