Rapid detection of α-thalassaemia alleles of --(SEA)/, -α(3.7)/ and -α(4.2)/ using a dual labelling, self-quenching hybridization probe/melting curve analysis

Abstract

Objectives: The aim of the study was to set up an alternative automatic molecular diagnostic method for deletional α-thalassaemia mutations without gel electrophoresis.

Methods: Based on the sequence variation within the two Z boxes and melting curve analysis of dually labelled probes, a real-time PCR assay was developed and validated for the rapid detection of major α-genotypes (--(SEA)/αα, --(SEA)/-α(3.7), --(SEA)/-α(4.2), --(SEA)/--(SEA), -α(3.7)/-α(3.7) and -α(4.2)/-α(4.2)).

Results: Samples with the -α(3.7)/-α(3.7), -α(4.2)/-α(4.2), --(SEA)/αα, --(SEA)/-α(3.7), --(SEA)/-α(4.2), and --(SEA)/--(SEA) genotypes could be clearly distinguished. The accuracy of this technique for these samples was 100% sensitivity and specificity.

Conclusion: This technique is rapid and reliable, demonstrating feasibility for use in large-scale population screening and prenatal diagnosis of deletional Hb H disease and Hb Bart's hydrops fetalis.

Keywords: Deletion; Dually labelled probes; Melting curve; α-Thalassaemia.