Detection of Hb Constant Spring, Hb Pakse and Hb Quong Sze by Real-Time PCR and High Resolution Melting Analysis
Abstract
Alpha thalassemia is one of the most common inherited anemia disorders in Thailand with prevalence at 30 to 40%. Compound heterozygous alpha-thalassemia 1 and non-deletional alpha-thalassemia gives rise to severe Hb H (--/-αT) disease. Thus, the rapid and accurate method for detection of non-deletional alpha-thalassemia is important for thalassemia treatment and prevention. The aim of this study was to develop HRM method for detection of non-deletional alpha-thalassemia including Hb CS, Hb PS and Hb QS. Two HRM primers, which specified for alpha-thalassemia 2, were used to identify three non-deletional alpha-thalassemia, and the optimized temperature was performed by gradient PCR. Next, real-time PCR and HRM analysis were established with positive control DNA carried Hb CS, HB PS, Hb QS and normal that were determined by DNA sequencing technique. After that, we examined 40 DNA samples from Thalassemia Unit, University of Phayao. The real-time PCR and HRM analysis could be able to identify control DNA including heterozygous Hb CS, Hb H CS, homozygous Hb CS, heterozygous Hb PS and homozygous Hb QS from negative DNA. 30 of 40 samples were negative, 3 samples were heterozygous Hb CS while, PCR product was not detected in 7 samples, that were the alpha-thalassemia 2 with 3.7 deletion. In conclusion, the real-time PCR and HRM method appears to be an accurate and sensitive method for the rapid screening and identification of Hb CS, Hb PS and Hb QS. Keywords : Alpha thalassemia; Hemoglobin H; High resolution melting analysis; HemoglobinReferences
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Nittayaboon, K., & Nopparatana, C. (2018). Molecular characterization of Hb H disease in southern Thailand. International Journal of Hematology, 108, 384-389.
Sanguansermsri, T. (2014). Condition of thalassemia trait. Journal of Hematology and Transfusion Medicine, 24(4), 329-332. (in Thai)
Seeratanachot, T., Shimbhu, D., Charoenkwan, P., & Sanguansermsri, T. (2015). Detection of deletion α+-thalassemia mutation [-α (3.7), -α (4.2)] by quantitative PCR assay. The Southeast Asian journal of tropical medicine and public health, 46(1), 110-115.
Suwannakhon, N., Seeratanachot, T., Mahingsa, K., Namwong, P., & Sanguansermsri, T. (2014). Prevalence of alpha thalassemia trait in the volunteered personals of Phayao University. Journal of Hematology and Transfusion Medicine, 24(2), 129-136.
Wee, Y. C., Tan, K. L., Chua, K. H., George, E., & Tan, J. A. (2009). Molecular characterisation of Haemoglobin Constant Spring and Haemoglobin Quong Sze with a Combine-Amplification Refractory Mutation System. The Malaysian journal of medical sciences: MJMS, 16(3), 21–28.
Lemmens-Zygulska, M., Eigel, A., Helbig, B., Sanguansermsri, T., Horst, J., & Flatz, G. (1996). Prevalence of alpha-thalassemias in northern Thailand. Human genetics, 98(3), 345–347.
Na-Nakorn, S., & Wasi, P. (1970). Alpha-thalassemia in Northern Thailand. American journal of human genetics, 22(6), 645–651.
Nittayaboon, K., & Nopparatana, C. (2018). Molecular characterization of Hb H disease in southern Thailand. International Journal of Hematology, 108, 384-389.
Sanguansermsri, T. (2014). Condition of thalassemia trait. Journal of Hematology and Transfusion Medicine, 24(4), 329-332. (in Thai)
Seeratanachot, T., Shimbhu, D., Charoenkwan, P., & Sanguansermsri, T. (2015). Detection of deletion α+-thalassemia mutation [-α (3.7), -α (4.2)] by quantitative PCR assay. The Southeast Asian journal of tropical medicine and public health, 46(1), 110-115.
Suwannakhon, N., Seeratanachot, T., Mahingsa, K., Namwong, P., & Sanguansermsri, T. (2014). Prevalence of alpha thalassemia trait in the volunteered personals of Phayao University. Journal of Hematology and Transfusion Medicine, 24(2), 129-136.
Wee, Y. C., Tan, K. L., Chua, K. H., George, E., & Tan, J. A. (2009). Molecular characterisation of Haemoglobin Constant Spring and Haemoglobin Quong Sze with a Combine-Amplification Refractory Mutation System. The Malaysian journal of medical sciences: MJMS, 16(3), 21–28.
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2022-05-18
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บทความวิจัย จากงานประชุมวิชาการระดับชาติ "วิทยาศาสตร์วิจัย ครั้งที่ 12"
