A novel gap-PCR with high resolution melting analysis for the detection of α-thalassaemia Southeast Asian and Filipino β 0-thalassaemia deletion

Abstract

Homozygosity for the α-thalassaemia Southeast Asian (α-SEA) and Filipino β 0-thalassaemia (β-FIL) deletions can cause serious complications leading to foetal death or life-long blood transfusions. A rapid and accurate molecular detection assay is essential in populations where the deletions are common. In this study, gap-polymerase chain reaction (PCR) with high resolution melting (HRM) analysis was developed to detect both the large deletions. Melting curves at 86.9±0.1°C were generated by normal individuals without the α-SEA deletion, 84.7±0.1°C by homozygous α-SEA deletion individuals and two melting curves at 84.7±0.1°C and 86.9±0.1°C by α-SEA deletion carriers. Normal individuals without the β-FIL deletion produce amplicons with a melting temperature (Tm) at 74.6±0.1°C, homozygous β-FIL individuals produce amplicons with Tm at 73.6±0.1°C and heterozygous β-FIL individuals generate two amplicons with Tm at 73.6±0.1°C and 74.6±0.1°C. Evaluation using blinded tests on 220 DNA samples showed 100% sensitivity and specificity. The developed assays are sensitive and specific for rapid molecular and prenatal diagnosis for the α-SEA and β-FIL deletions.