基于实时荧光PCR的探针熔解曲线分析技术和反向点杂交技术应用于β-地中海贫血基因诊断与产前诊断的对比研究  

Comparison between probe melting curve analysis based on real-time fluorescent PCR and reverse dot blot assay for gene and prenatal diagnosis of β-thalassemia

作　　者：肖奇志[1] 周玉球[1] 谢建红[1] 张永良[1] 

机构地区：[1]珠海市妇幼保健院检验科珠海市医学遗传研究所,519001

出　　处：《中华检验医学杂志》2012年第5期413-417,共5页Chinese Journal of Laboratory Medicine

基　　金：广东省科技计划项目资助课题（2009A030301002）;广东省自然科学基金资助项目（04101691）

摘　　要：目的评价基于实时荧光PCR的探针熔解曲线分析（PMCA）技术作为β-地中海贫血（β-地贫）基因诊断和产前诊断方法的可行性。方法收集2008至2010年在珠海市妇幼保健院经过血液学表型筛查初步诊断为轻型β-地贫的血标本119份、重型β-地贫血标本4份和正常血标本12份。此外，收集严重类型β-地贫高风险孕妇的羊水标本44份和脐带血标本8份。采用双盲法，采用PMCA技术和反向点杂交（RDB）技术对上述187份临床标本分别进行β-地贫基因突变检测，并以DNA测序技术作为金标准，对上述两种方法检测B-地贫基因突变的检测性能进行评估。结果探针熔解曲线分析法除了漏检1例Ini（ATG〉AGG）／N和1例不能区分为CD43（G〉T）／N还是CD37（G〉A）／N外，其余的185份标本的β-地贫基因型均能正确检出；而RDB法有1例CD71-72（＋T）／N未正确检出。以DNA测序技术作为金标准，探针熔解曲线分析法检测的敏感度、特异度、阴性预测值、阳性预测值和诊断效率分别为98．75％、100．00％、93．10％、100．00％、98．93％，RDB法分别为99．38％、100．00％、96．42％、100．00％、99．47％；两者间诊断效率的差异无统计学意义（P=0．999）。且产前诊断的结果与胎儿出生或引产后血样的基因诊断或产后随访结果完全一致。结论PMCA技术可作为RDB技术检测中国人群β-地贫基因突变的替代或验证方法。Objective To evaluate the reliability of the probe melting curve analysis （PMCA） based on real-time fluorescent PCR assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia. Methods A total of 135 cases of peripheral blood samples were collected from Zhuhai Municipal Maternity and Child Healthcare Hospital between 2008 and 2010. All the samples were performed preliminary diagnosis according to the hematological data. Of these, 119 cases were diagnosed as β-thalassemia trait, 4 cases were diagnosed as severe thalassemia and 12 cases were normal. In addition, 44 cases of amniotic fluid and 8 cases of cord blood with high-risk for severe β-thalassemia were also collected. The diagnostic reliability of the PMCA assay and reverse dot blot assay was evaluated on 187 above-mentioned cases by direct DNA sequencing analysis in a double-blind study. Results The genotypes of 185 cases were detected accurately based on the PMCA assay except for two cases：one heterozygote with Ini （ATG 〉 AGG） was omitted and another heterozygote couldn＇t be distinguished between CD43 （G 〉 T） and CD37 （G 〉 A）. For the RDB assay, only one heterozygote with CD71-72 （ ＋ T） was not detected accurately in the above-mentioned cases. Compared with the DNA sequencing analysis, the sensitivity, specificity, negative predictive value, positive predictive value and diagnostic efficiency of the PMCA assay were 98. 75%, 100. 00% , 93.10%,100. 00% and 98. 93% , respectively. The corresponding value of the RDB assay were 99. 38% , 100. 00% , 96.42%, 100. 00% and 99.47%, respectively. There were no significant between-group differences in the diagnostic efficiency of the two assays （ P 〉 0. 05 ） . The results of prenatal diagnosis were in complete concordance with the follow up results, after the birth or induced labour of the fetuses. Conclusions The PMCA assay can be used as an alternative and verified method of RDB assay for the genetic diagnosis and prenatal diagnosis of β-thalassemia.

关 键 词：Β地中海贫血 聚合酶链反应 熔解曲线分析 核酸杂交 产前诊断 

分 类 号：R440[医药卫生—诊断学] R714.5[医药卫生—临床医学]