SYTO9 and SYBR Green1 with High Resolution Melting Analysis for Molecular Confirmatory Testing of the Common Southeast Asian β⁰-thalassemia Mutations

Abstract

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 β⁰-thalassemia (β⁰-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 A₂ levels between 3.1 and 9.9%. The SYTO9 HRM analysis showed specific characteristic peaks of each investigated type of β⁰-thal mutation. Results of MARMS-PCR followed by SYTO9 HRM analyses were completely consistent with results of those analyzed by gel electrophoresis. Moreover, the β⁰-thal 3.5 kb gene deletion could be identified in two samples with Hb A₂ 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 β-thal.

Keywords:

• β-Thalassemia (β-thal)
• Hb A₂
• High resolution melting (HRM) analysis
• SYTO9
• SYBR GREEN1

ACKNOWLEDGMENTS

The authors thank technicians in the Health Promoting Hospital Chiang-Mai, Chiang-Mai, Thailand for their help and assistance. We are grateful to Gerald W. Rock (retired from the United States Department of the Treasury, Washington, D.C., USA), Kallayanee Treesuwan (laboratory coordinator of the Health Concepts International Ltd., Bangkok, Thailand) and Dr. Pranee Leechanachai (Faculty of Associated Medical Sciences, Chiang-Mai University, Chiang-Mai, Thailand) for editing the manuscript. This study has been supported in part by Q Bioscience Ltd., Bangkok, Thailand.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.