Publication: Rapid diagnosis of α-thalassemia by melting curve analysis
Issued Date
2010-05-01
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ISSN
15251578
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2-s2.0-77951734486
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Mahidol University
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SCOPUS
Bibliographic Citation
Journal of Molecular Diagnostics. Vol.12, No.3 (2010), 354-358
Suggested Citation
Thongperm Munkongdee, Phantip Vattanaviboon, Parichut Thummarati, Paijit Sewamart, Pranee Winichagoon, Suthat Fucharoen, Saovaros Svasti Rapid diagnosis of α-thalassemia by melting curve analysis. Journal of Molecular Diagnostics. Vol.12, No.3 (2010), 354-358. doi:10.2353/jmoldx.2010.090136 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/28717
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Title
Rapid diagnosis of α-thalassemia by melting curve analysis
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Abstract
α-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of α-globin protein. Couples who both carry the α-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the α-thalassemia-1 gene is the most effective strategy to prevent and control this severe form of thalassemia. In this study, a new and accurate method for α-thalassemia diagnosis was developed by genotyping α-thalassemia-1, the Southeast Asian type (--SEA) and Thai type (--THAI) deletions, using multiplex PCR followed by a melting curve analysis. Primers were designed to specifically amplify two deletion fragments, the --SEAand --THAIdeletions and two normal fragments, ψζ- and α2-globin gene. The primers were capable of distinguishing α-thalassemia 1 heterozygotes from α-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the --THAI, --SEA, ψζ-globin, and α2-globin gene fragments were 79.9 ± 0.2, 89.4 ± 0.5, 92.8 ± 0.2, and 85.0 ± 0.2°C, respectively. Melting curve analysis was performed in 130 subjects in parallel with conventional gap-PCR analysis, and results showed 100% concordance. This method eliminates the post-PCR electrophoresis process, which is laborious, and allows high throughput screening suitable for large population screening for prevention and control of thalassemia. Copyright © American Society for Investigative Pathology and the Association for Molecular Pathology.