A Melting Curve Analysis-Based PCR Assay for One-Step Genotyping of β-Thalassemia Mutations A Multicenter Validation

The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis based genotyping assay (MeltPro HBB assay) for 24 beta-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of beta-thalassemia minor or major, using a double-blind analysis in a multicenter validation study. Reproducibility of the assay was 100%, and the limit of detection was 10 pg per reaction. All 24 beta-thalassemia mutations were accurately genotyped, and beta-thalassemia genotypes were correctly determined in all 1022 samples, yielding overall sensitivity and specificity of 100%. The concordance rate was 99.4% between this assay and the reference method. It was concluded that the MeltPro HBB assay is useful for reliable genotyping of multiple beta-thalassemia mutations in clinical settings and may have potential as a versatile method for rapid genotyping of known mutations because of its high throughput, accuracy, ease of use, and low cost. (J mol Diagn 2011, 13:427-435; DOI: 10.1016/j.jmoldx.2011.03.005)