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Updated: Apr 4, 2026
Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
Published on: November 10, 2015
Rapid detection of non-deletional mutations causing α-thalassemia by multicolor melting curve analysis
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.
