Korean J Lab Med.  2011 Jul;31(3):138-142. 10.3343/kjlm.2011.31.3.138.

Detection of alpha-thalassemia-1 Southeast Asian and Thai Type Deletions and beta-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis

• Pornprasert S
• Wiengkum T
• Srithep S
• Chainoi I
• Singboottra P
• Wongwiwatthananukit S

• KMID: 1735843
• DOI: http://doi.org/10.3343/kjlm.2011.31.3.138

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 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.

Keyword

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

MeSH Terms

Asia, Southeastern
Asian Continental Ancestry Group/*genetics
*Gene Deletion
Genotype
Humans
Organic Chemicals/*chemistry
Phase Transition
Polymerase Chain Reaction/*methods
Reagent Kits, Diagnostic
Thailand
Transition Temperature
alpha-Thalassemia/diagnosis/*genetics
beta-Thalassemia/diagnosis/*genetics

Figure

• 

  Fig. 1 Representative results of dissociation curve analysis for α-thalassemia-1 SEA and Thai type deletions and β-thalassemia 3.5-kb deletion. DNA samples were obtained from 5 normal individuals, 5 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 deletions.Abbreviations: dF/dT, derivative of the fluorescence/derivative of the temperature; SEA, Southeast Asian.

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