passages.id: 36
passages.type: title
passages.text: Detection of a-thalassemia-1 Southeast Asian and Thai type deletions and b-thalassemia 3.5-kb deletion by single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting analysis.
passages.id: 37
passages.type: abstract
passages.text: BACKGROUND: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. METHODS: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of a-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and b-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with a-thalassemia-1 SEA type deletion, 2 with a-thalassemia-1 Thai type deletion, and 2 with heterozygous b-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from a-thalassemia-1 SEA type deletion, a-thalassemia-1 Thai type deletion, b-thalassemia 3.5-kb gene deletion, and the wild-type b-globin gene had specific peak heights at mean melting temperature (T(m)) values of 86.89   , 85.66   , 77.24   , and 74.92   , respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. CONCLUSIONS: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of a- and b-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.