A novel gap-PCR with high resolution melting analysis for the detection of alpha-thalassaemia Southeast Asian and Filipino beta(o)-thalassaemia deletion

作者：Kho Siew Leng; Chua Kek Heng; George Elizabeth; Tan Jin Ai Mary Anne^*

来源：Scientific Reports, 2015, 5(1): 13937.

DOI：10.1038/srep13937

Homozygosity for the alpha-thalassaemia Southeast Asian (alpha-SEA) and Filipino beta(o)-thalassaemia (beta-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 degrees C were generated by normal individuals without the alpha-SEA deletion, 84.7 +/- 0.1 degrees C by homozygous alpha-SEA deletion individuals and two melting curves at 84.7 +/- 0.1 degrees C and 86.9 +/- 0.1 degrees C by alpha-SEA deletion carriers. Normal individuals without the beta-FIL deletion produce amplicons with a melting temperature (Tm) at 74.6 +/- 0.1 degrees C, homozygous beta-FIL individuals produce amplicons with Tm at 73.6 +/- 0.1 degrees C and heterozygous beta-FIL individuals generate two amplicons with Tm at 73.6 +/- 0.1 degrees C and 74.6 +/- 0.1 degrees 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 alpha-SEA and beta-FIL deletions.

• 出版日期2015-9-14