RESUMO
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 alpha-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and beta-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 alpha-thalassemia-1 SEA type deletion, 2 with alpha-thalassemia-1 Thai type deletion, and 2 with heterozygous beta-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from alpha-thalassemia-1 SEA type deletion, alpha-thalassemia-1 Thai type deletion, beta-thalassemia 3.5-kb gene deletion, and the wild-type beta-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89degrees C, 85.66degrees C, 77.24degrees C, and 74.92degrees C, 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 alpha- and beta-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.
Assuntos
Humanos , Sudeste Asiático , Povo Asiático/genética , Deleção de Genes , Genótipo , Compostos Orgânicos/química , Transição de Fase , Reação em Cadeia da Polimerase/métodos , Kit de Reagentes para Diagnóstico , Tailândia , Temperatura de Transição , Talassemia alfa/diagnóstico , Talassemia beta/diagnósticoRESUMO
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 alpha-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and beta-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 alpha-thalassemia-1 SEA type deletion, 2 with alpha-thalassemia-1 Thai type deletion, and 2 with heterozygous beta-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from alpha-thalassemia-1 SEA type deletion, alpha-thalassemia-1 Thai type deletion, beta-thalassemia 3.5-kb gene deletion, and the wild-type beta-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89degrees C, 85.66degrees C, 77.24degrees C, and 74.92degrees C, 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 alpha- and beta-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.