Rapid diagnosis of alpha-thalassemia by melting curve analysis.
alpha-Thalassemia is an inherited hemoglobin disorder that results from defective synthesis of alpha-globin protein. Couples who both carry the alpha-thalassemia-1 gene are at risk of having a fetus with Hb Bart's hydrops fetalis. Rapid and accurate screening for individuals carrying the alpha-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 alpha-thalassemia diagnosis was developed by genotyping alpha-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 --(SEA) and --(THAI) deletions and two normal fragments, psizeta- and alpha2-globin gene. The primers were capable of distinguishing alpha-thalassemia 1 heterozygotes from alpha-thalassemia 2 homozygotes, which are unable to be diagnosed by standard hematological data and hemoglobin typing. The melting temperatures of the --(THAI), --(SEA), psizeta-globin, and alpha2-globin gene fragments were 79.9 +/- 0.2, 89.4 +/- 0.5, 92.8 +/- 0.2, and 85.0 +/- 0.2 degrees 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.
Munkongdee
Thongperm
T
Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthon 4 Rd., Phuttamonthon, Nakornpathom 73170, Thailand.
Vattanaviboon
Phantip
P
Thummarati
Parichut
P
Sewamart
Paijit
P
Winichagoon
Pranee
P
Fucharoen
Suthat
S
Svasti
Saovaros
S
D005838:Genotype; D006454:Hemoglobins / Q000235:genetics; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics*
D006454:Hemoglobins