Aims Alpha-thalassaemia is one of the most common monogenic disorders worldwide. Due to high guanine-cytosine (GC) content and high mutation diversity in α-globin gene cluster, deletional and non-deletional mutations were usually separately detected with different methods. The aim of this study was to develop a novel one-step method for α-thalassaemia genotyping.
Methods A multiplex symmetric PCR melting curve strategy was designed for one-step α-thalassaemia genotyping. Based on this strategy, a novel method was developed to simultaneously detect four common deletional (-α3.7 , -α4.2 , _ _SEA , --THAI ) and five common non-deletional (αCD30(-GAG)α, αCD31(G>A)α, αWSα, αQSα, αCSα) α-thalassaemia mutations in a closed-tube reaction. This method was also evaluated by double-blind detection of 235 genotype-known samples and 1630 clinical samples.
Results All nine α-thalassaemia mutations could be accurately identified by this novel method within 3 hours. The evaluation results also showed a 100% concordance with comparison methods.
Conclusions This method is rapid, accurate, low-cost and easy to operate, which can be used for molecular screening and genetic diagnosis of α-thalassaemia in clinical practice. The multiplex symmetric PCR melting curve strategy designed in this study can also provide an effective approach to the method development for high GC content templates and multiple mutations.
Data availability statement
Data are available on reasonable request. All data relevant to the study are included in the article or uploaded as online supplemental information.
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Handling editor Runjan Chetty.
JQ and JH contributed equally.
Contributors WZ is the guarantor of this study. WZ and JH supervised this study and revised the manuscript. JQ conducted the experiments and wrote the original draft. NL and QQ performed statistical analyses of the results. YL and FT drafted the manuscript. PZ and WG collected samples. All authors approved the manuscript.
Funding This study was supported by the National Natural Science Foundation of China (81972008), Basic and Applied Basic ResearchFoundation of Guangdong Province (2021A1515012585).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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