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.

A method for detection of hemoglobin (Hb) E mutation was developed based on allelic discrimination analysis. Two probes labeled with different fluorescent reporter dyes were designed to specifically detect variation of a single nucleic acid polymorphism (SNP) site in the target template sequence. Polymerase chain reaction (PCR) products of normal allele and mutant allele were detected directly by analyzing fluorescent signal of each probe. This method was validated in term of accuracy (by comparing with sequence analysis) and reproducibility. The % CV between run precision was 5.16-8.86%. The narrow scatter of the results confirmed the reproducibility of the assay. This technique is a rapid, reliable and cost-effective method to differentiate Hb E homozygosity from beta(0)-thalassemia/Hb E in populations with a high frequency of beta-thalassemia and Hb E.

Fluorescence-based DNA sequence analysis was developed for identification of beta-globin gene mutations in the Thai population. The beta-globin gene was directly sequenced in two runs and the sequencing electropherogram allowed unambiguous detection of nucleotide substitutions, frameshifts, and small insertions/deletions in heterozygote and homozygote. The method was validated and successfully applied in routine analysis of 416 individuals with beta-thalassemia disease, beta-thalassemia/hemoglobin (Hb) E and Hb variants. Twenty-five different beta-globin gene mutations were identified. Two Hb variants, Hb Tacoma [codon 30 (G-T)] and Hb Tende [codon 124 (C-T)], were also identified for the first time in a Thai population. Automated fluorescence-based DNA sequence analysis provides a rapid and reliable method for identification of common, rare and unknown beta-globin gene mutations, which is essential for prevention and control of thalassemia and hemoglobinopathy in Thailand.