SYTO9 and SYBR GREEN1 with high resolution melting analysis for molecular confirmatory testing of the common Southeast Asian beta(0)-thalassemia mutations

Gel electrophoresis and ethidium bromide staining are routine methods in molecular laboratories. However, they are not ideally suited to large scale analyses in clinical laboratories. We used SYTO9 and high resolution melting (HRM) analyses for identification of the common beta(0)-thalassemia (beta(0)-thal) in Southeast Asia including the codons 17 (A>T), 41/42 (-TCTT) and 71/72 (+A) mutations. Multiplex amplification refractory mutation system-polymerase chain reaction (MARMS-PCR) was performed on 102 blood samples that had Hb A(2) levels between 3.1 and 9.9%. The SYTO9 HRM analysis showed specific characteristic peaks of each investigated type of beta(0)-thal mutation. Results of MARMS-PCR followed by SYTO9 HRM analyses were completely consistent with results of those analyzed by gel electrophoresis. Moreover, the beta(0)-thal 3.5 kb gene deletion could be identified in two samples with Hb A(2) levels of >4% by GREEN1 HRM analysis. The SYTO9 and SYBR GREEN1 HRM analysis is simple and rapid. This approach will facilitate laboratory diagnosis and genetic counseling for regions with a high prevalence of beta-thal.