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Korean J Lab Med. 2011 Jul;31(3):138-42. doi: 10.3343/kjlm.2011.31.3.138. Epub 2011 Jun 28.

Detection of α-thalassemia-1 Southeast Asian and Thai type deletions and β-thalassemia 3.5-kb deletion by single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting analysis.

Author information

1
Department of Medical Technology, Chiang-Mai University, Chiang-Mai, Thailand. sakornmi001@gmail.com

Abstract

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 α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-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 α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion.

RESULTS:

HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-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 α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

KEYWORDS:

High-resolution melting analysis; Multiplex real-time PCR; SYBR Green1; α-thalassemia-1; β-thalassemia 3.5-kb deletion

PMID:
21779184
PMCID:
PMC3129341
DOI:
10.3343/kjlm.2011.31.3.138
[Indexed for MEDLINE]
Free PMC Article

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