One-step genotyping of α-thalassaemia by multiplex symmetric PCR melting curve

Aims Alpha-thalassaemia is one of the most common monogenic disorders worldwide. Due to high guanine-cytosine (GC) content and high mutation diversity in alpha-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 alpha-thalassaemia genotyping. Methods A multiplex symmetric PCR melting curve strategy was designed for one-step alpha-thalassaemia genotyping. Based on this strategy, a novel method was developed to simultaneously detect four common deletional (-alpha(3.7) , -alpha(4.2) , (_ _SEA) , --(THAI) ) and five common non-deletional (alpha(CD30(-GAG))alpha, alpha(CD31(G>A))alpha, alpha(WS)alpha, alpha(QS)alpha, alpha(CS)alpha) alpha-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 alpha-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 alpha-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.