A rapid DNA-based assay is essential for clinical diagnosis and mass screening in thalassemia-prevention programs. Because of high homology and guanine-cytosine–rich and complex second structure of α-globin genes, it is rather difficult to develop a feasible and simple method for α-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 α-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 α-thalassemia mutations in clinical practice. Especially, the strategy may have the potential to be a versatile scheme for rapid genotyping of other gene mutations because of its high throughput, sufficient stability, low cost, and simple operation.

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-珠蛋白基因的高度同源性和富含鸟嘌呤胞嘧啶且复杂的二级结构，因此很难开发一种可行且简单的α-地贫基因分型方法。在本研究中，设计了嵌套不对称 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。在此，一种用于非缺失性 α-地贫突变基因分型的新型一步分析法，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 CD31 ( HBA2 : c.95G>A ) 在单个封闭管中被建立和评估。在对 255 个已知基因型样本和 1250 个临床样本的盲分析中，所有 5 种突变均以 100% 的一致率准确确定。总之，该测定可用于临床实践中非缺失性 α-地贫突变的快速和可靠的基因分型。特别是，该策略具有高通量、足够的稳定性、低成本和简单的操作等优点，有可​​能成为其他基因突变快速基因分型的通用方案。