A Nested Asymmetric PCR Melting Curve Assay for One-Step Genotyping of Nondeletional α-Thalassemia Mutations
A Nested Asymmetric PCR Melting Curve Assay for One-Step Genotyping of Nondeletional α-Thalassemia Mutations
复制标题
DOI：
10.1016/j.jmoldx.2020.03.002
复制
发表时间：
2020-06-01
影响因子：
4.1
通讯作者：
Zhou, Wanjun
中科院分区：
文献类型：
--
作者：
Qin, Jiachun;Xu, Mingli;Zhou, Wanjun
基于 DNA 的快速检测对于地中海贫血预防计划中的临床诊断和大规模筛查至关重要。由于α珠蛋白基因同源性高、鸟嘌呤丰富、二级结构复杂，开发一种可行、简单的α地中海贫血基因分型方法相当困难。在本研究中，设计了一种巢式不对称PCR熔解曲线分析策略来解决这些因素并确保灵敏度和准确性。本文中，一种用于非缺失性α地中海贫血突变基因分型的新型一步测定法，包括血红蛋白 (Hb) Westmead (HBA2: c.369C>G)、Hb Quong Sze (HBA2: c.377T>C)、Hb Constant Spring (HBA2: c.427T>C)、CD30 (HBA2: c.91-93delGAG) 和在单个封闭管中建立并评估 CD31 (HBA2: c.95G>A)。通过对 255 个基因型已知样本和 1250 个临床样本的盲分析，所有 5 种突变均得到准确测定，一致率为 100%。总之，该测定对于临床实践中非缺失性α-地中海贫血突变的快速、可靠的基因分型是有用的。特别是，由于其高通量、足够的稳定性、低成本和操作简单，该策略可能有潜力成为其他基因突变快速基因分型的通用方案。
A rapid DNA-based assay is essential for clinical diagnosis and mass screening in thalassemia-prevention programs. Because of high homology and guanine-cytosineerich and complex second structure of alpha-globin genes, it is rather difficult to develop a feasible and simple method for alpha-thalassemia genotyping. In this study, a strategy of nested asymmetric PCR melting curve analysis was designed to tackle these factors and ensure sensitivity and accuracy. Herein, a novel one-step assay for genotyping of nondeletional alpha-thalassemia mutations, including hemoglobin (Hb) Westmead (HBA2: c.369C>G), Hb Quong Sze (HBA2: c.377T>C), Hb Constant Spring (HBA2: c.427T>C), CD30 (HBA2: c.91-93delGAG), and CD31 (HBA2: c.95G>A) in a single closed tube, was established and evaluated. All five mutations were accurately determined with the concordance rate of 100% in a blind analysis of 255 genotype-known samples and 1250 clinical samples. In conclusion, this assay is useful for rapid and reliable genotyping of nondeletional alpha-thalassemia mutations in clinical practice. Especially, the strategy may have the potential to be a versatile scheme for rapid genotyping of other genemutations because of its high throughput, sufficient stability, low cost, and simple operation.