Abstract
α-Thalassemia, caused by mutations in the α-globin genes, is one of the most common monogenic inherited disorders in the world. However, non-deletional α-thalassemia mutations remain undetected in routine clinical testing due to the lack of a suitable method. In this study, a closed- and single-tube assay for the detection of six common non-deletional α-thalassemia mutations in the HBA2 gene was developed based on multicolor melting curve analysis. The assay consisted of one pair of primers specific for the HBA2 gene and four dual-labeled, self-quenched probes targeting six non-deletional α-thalassemia mutations. The sensitivity, reproducibility, and accuracy of the method were validated via 700 genomic DNA samples. The assay had a reproducibility of 100%, could detect gDNA of different genotype as low as 1 ng per reaction, and had an overall accuracy of 100% when compared with RDB analysis and Sanger sequencing. The developed assay is rapid, robust, and cost-effective while maintaining high sensitivity, specificity, and throughput.
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Citations
Feb 23, 2017·Scientific Reports·Xudong WangQingge Li
May 1, 2018·BioMed Research International·Chao WangDaru Lu
Aug 14, 2020·Hemoglobin·Manit Nuinoon, Dararat Horpet
Nov 6, 2020·Hemoglobin·Ai-Ping JuDong-Zhi Li
May 25, 2021·Frontiers in Pediatrics·Qinlong ZengTong Ou