目的:建立一种基于两对交叉引物PCR技术(PCR with confronting two-pair primers,PCR-CTPP)同时快速检测非缺失型α地中海贫血WS、QS和CS三种突变类型的方法及其在临床中的应用.方法:根据α珠蛋白基因中发生WS、QS和CS突变的区域基因序列,分别针对单个位点设计两对引物即通用外引物和特异性引物,经过聚合酶链反应、电泳鉴别基因型,同时检测非缺失型α地中海贫血.将该方法与PCR反向斑点杂交(PCR-RDB)技术比较,验证其检测准确性.结果:PCR-CTPP方法可用于准确检测非缺失型α地中海贫血的WS、QS和CS突变类型,结果显示与常规非缺失型α地中海贫血PCR-RDB检测方法一致.结论:PCR-CTPP技术检测非缺失型α地中海贫血WS、QS、CS三种突变位点,简单快速、经济省力,可用于非缺失型α地中海贫血的快速筛查和分子流行病学研究,适合在一般实验室推广应用.适用于非缺失型α地中海贫血的人群筛查及婚前或产前筛查,适合各个层次医院应用.
Establishment and clinical application of PCR-CTPP method for detection of non-deletion alpha thalassemia point mutations
Objective:To establish a method for the simultaneous rapid detection of three mutation types,WS,QS,and CS,in non-deletion alpha thalassemia based on PCR with confronting two-pair primers(PCR-CT-PP)and its application in clinical practice.Methods:Based on the gene sequences of the region of the α pearl pro-tein gene where WS,QS,and CS mutations occur,two pairs of primers,universal external primers and specific primers,were designed for a single locus,respectively,and the genotypes were identified by a polymerase chain reaction and electrophoresis to detect the non-deletion type of α thalassemia at the same time.The accuracy of the method was verified by comparing it with PCR-RDB.Results:The PCR-CTPP method can be used to accurately detect the WS,QS,and CS mutation types in non-deletion α thalassemia,and the results showed agreement with the conventional PCR-RDB assay for non-deletion α thalassemia.Conclusion:PCR-CTPP is a simple,rapid,and cost-effective technique for the detection of WS,QS,and CS mutation sites in non-deficiency α thalassemia,which can be used for rapid screening and molecular epidemiological studies of non-deficiency α thalassemia,and it is suitable for general laboratory applications.It is suitable for population screening for non-deletion alpha-thalasse-mia and premarital or prenatal screening and is suitable for application in hospitals at all levels.
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