Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR-Based Multicolor Melting Curve Analysis
Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR-Based Multicolor Melting Curve Analysis
复制标题
通过基于实时 PCR 的多色熔解曲线分析同时对 α-地中海贫血缺失和非缺失突变进行基因分型
DOI：
10.1016/j.jmoldx.2017.04.003
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发表时间：
2017-07-01
影响因子：
4.1
通讯作者：
Li, Qingge
中科院分区：
文献类型：
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作者：
Huang, Qiuying;Wang, Xudong;Li, Qingge
由血红蛋白α-珠蛋白链合成缺陷引起的α-地中海贫血是最常见的遗传隐性血红蛋白异常。由于α-珠蛋白基因簇的各种大缺失和非缺失突变，缺陷α-珠蛋白基因的遗传检测是具有挑战性的。通常需要使用复杂且容易出错的开管方法对它们进行单独检测。我们报告了一种新的实时PCR检测方法，可以同时基因型的四个主要deletionat和三个常见的非缺失突变在两个平行的反应，通过使用熔解曲线分析。该封闭管测定的周转时间在3.5小时内，检测限为每次反应5 ng人类基因组DNA，并且在镶嵌样品中可以检测到低至5%的突变体DNA。使用1213个预先表征的基因组DNA样品以双盲方式评价该测定。所有七种α地中海贫血突变均得到准确的基因分型，与比较分析的一致性为99.3%。14个不一致的样本含有HK α α等位基因，未被检测到。传统的方法。考虑到其快速性、易用性和准确性，我们得出结论，我们的实时PCR检测可推荐作为α-地中海贫血的替代筛查和诊断工具。
alpha-Thalassemia, which is caused by defective synthesis of the hemoglobin alpha-globin chains, is the most commonly inherited recessive hemoglobin abnormality. Genetic detection of a defective alpha-globin gene is challenging because of a variety of large deletions of the alpha-globin gene cluster and nondeletional mutations. Separate detections of them are often required using complex and error prone open-tube methods. We report a novel real-time PCR-based assay that can simultaneously genotype four major deletionat and three common nondeletional mutations in two parallel reactions by using multicolor melting curve analysis. The turnaround time of this closed-tube assay was within 3.5 hours, the limit of detection was 5 ng of human genomic DNA per reaction, and as low as 5% mutant DNA could be detected in the mosaic samples. The assay was evaluated using 1213 pre-characterized genomic DNA samples in a double-blind manner. ALL seven alpha-thalassemia mutations were accurately genotyped, yielding a 99.3% concordance with the comparison assays. The 14 discordant samples contained the HK alpha alpha allele that was undetected by. the traditional methods. Considering its rapidity, ease of use, and accuracy, we concluded that our real-time PCR assay may be recommended as an alternative screening and diagnostic tool for alpha-thalassemia.