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