title: A novel gap-PCR with high resolution melting analysis for the detection of α-thalassaemia Southeast Asian and Filipino β°-thalassaemia deletion.
abstract: Homozygosity for the α-thalassaemia Southeast Asian (α-SEA) and Filipino β°-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.
authors.affiliation: Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Siew Leng
authors.initials: SL
authors.lastname: Kho
authors.affiliation: Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Kek Heng
authors.initials: KH
authors.lastname: Chua
authors.affiliation: Assunta Hospital, Petaling Jaya, Selangor, Malaysia.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: George
authors.affiliation: Department of Biomedical Science, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
authors.forename: Jin Ai Mary Anne
authors.initials: JA
authors.lastname: Tan
mesh_terms: D044466:Asian People; D004247:DNA / Q000032:analysis / Q000378:metabolism; D006801:Humans; D010679:Philippines; D060888:Real-Time Polymerase Chain Reaction; D044366:Transition Temperature; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D055544:beta-Globins; D004247:DNA