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.
Qiuying
Q
Huang
Xudong
X
Wang
Ning
N
Tang
Chunjiang
C
Zhu
Tizhen
T
Yan
Ping
P
Chen
Qingge
Q
Li
D004247:DNA / Q000737:chemistry; D005821:Genetic Techniques; D005838:Genotype; D006801:Humans; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013997:Time Factors; D044366:Transition Temperature; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics*
D004247:DNA