Accessible Requires Authentication Published by De Gruyter August 22, 2015

Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis

Qiuying Huang, Xudong Wang, Ning Tang, Chunjiang Zhu, Tizhen Yan, Ping Chen and Qingge Li

Abstract

Background:α-Thalassemia, caused by mutations in the α-globin genes, is one of the most common monogenic inherited disorders in the world. However, non-deletional α-thalassemia mutations remain undetected in routine clinical testing due to the lack of a suitable method. In this study, a closed- and single-tube assay for the detection of six common non-deletional α-thalassemia mutations in the HBA2 gene was developed based on multicolor melting curve analysis.

Methods: The assay consisted of one pair of primers specific for the HBA2 gene and four dual-labeled, self-quenched probes targeting six non-deletional α-thalassemia mutations. The sensitivity, reproducibility, and accuracy of the method were validated via 700 genomic DNA samples.

Results: The assay had a reproducibility of 100%, could detect gDNA of different genotype as low as 1 ng per reaction, and had an overall accuracy of 100% when compared with RDB analysis and Sanger sequencing.

Conclusions: The developed assay is rapid, robust, and cost-effective while maintaining high sensitivity, specificity, and throughput.


Corresponding authors: Ping Chen, Guangxi Key Laboratory of Thalassemia Research, Guangxi Zhuang Autonomous Region, Nanning 530021, P.R. China, E-mail: ; and Hemoglobin Laboratory, the First Affiliated Hospital of Guangxi Medical University, Guangxi Key Laboratory of Thalassemia Research, Guangxi Zhuang Autonomous Region, Nanning, P.R. China; and Qingge Li, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, P.R. China, E-mail: ; and State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Centre of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, P.R. China
aQiuying Huang and Xudong Wang contributed equally to this work.

Acknowledgments

We thank Dr. Ineke Rood for critical reading of the manuscript.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Research funding: This research work was supported by the National Natural Science Foundation of China (Grant No. 81101323), Guangxi Key Laboratory Project (Grant No. 12-071-05), Guangxi Scientific Research and Technology Development Project (Grant No. 1298003-4-1), and Liuzhou Scientific Research and Technology Development Projects (Grant No. 2014G020404).

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests:The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Supplemental Material:

The online version of this article (DOI: 10.1515/cclm-2015-0173) offers supplementary material, available to authorized users.

Received: 2015-2-19
Accepted: 2015-7-22
Published Online: 2015-8-22
Published in Print: 2016-3-1

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