Rapid diagnosis of alpha(o)-thalassemia using the relative quantitative PCR and the dissociation curve analysis

A method for diagnosis of alpha(o)-thalassemia was developed based on detection of accumulated PCR product using SYBR Green I, a double-stranded DNA binding dye, and a fluorescence-detecting thermocycler. Primers were designed to specifically amplify - -SEA and - -Thai deletions of alpha(o)-thalassemia. Albumin was selected as the reference gene. The comparative CT method was used to quantitate the target gene relative to the albumin gene. Dissociation curve analysis was used as a qualitative tool to distinguish different types of alpha(o)-thalassemia. In this study, the melting temperatures of the - -Thai and - -SEA deletions were 83 and 86 degrees C, respectively. Application of the assay for the diagnosis of alpha(o)-thalassemia heterozygotes and homozygotes is reported. The assay is highly robust and amenable to high throughput. It is thus a useful tool for the rapid detection of alpha(o)-thalassemia in populations with a high frequency of alpha(o)-thalassemia, - -SEA and - -Thai deletions.