Abstract:

Objective To establish and evaluate a method for determining 3 α-thalassemia non-deletional mutations（αWS，αQS，αCS） and 8 β-thalassemia point mutations [CD26（G>A），CD41/42（-TCTT）， nt29（A>G） ， IVS-Ⅱ-654（C>T）， nt28（A>G） ， CD71/72（+A） ，CD17（A>T）， CD27/28（+C）] simultaneously using hetero-tail dual-labeled fluorescent probe hydridization. Methods Polymerase chain reaction（PCR） primers and hetero-tail dual-labeled fluorescent probes targeting 11 most common α-thalassemia non-deletional and β-thalassemia point mutations were designed. An optimized multiplex asymmetric PCR and hetero-tail dual-labeled fluorescent probe hydridization were established. Performance specifications（accuracy，specificity and sensitivity） were evaluated by the national reference materials for thalassemia. Totally，200 clinical samples were determined by PCR-reverse dot blot kit and hetero-tail dual-labeled fluorescent probe hydridization concurrently to assess the consistency. Results For 11 mutations within the determination range，hetero-tail dual-labeled fluorescent probe hydridization showed 100% consistency with the national reference materials for thalassemia，and none of the mutations outside the determination range were determined. For different concentrations of Hb CS，CD41/42 and IVS-Ⅱ-654 reference materials ranging from 1.0，2.5，5.0 to 10.0 ng/μL，the results were consistent. The consistency rate between PCR-reverse dot blot kit and hetero-tail dual-labeled fluorescent probe hydridization was 100%. Conclusions Hetero-tail dual-labeled fluorescent probe hydridization enables fast and accurate determination of 11 α-thalassemia non-deletional and β-thalassemia point mutations simultaneously by one-step real-time PCR，which reduces turn-around time and contamination risk and shows potential for future clinical application.

Key words: Hetero-tail dual-labeled fluorescent probe, Multiplex asymmetric polymerase chain reaction, Non-deletional mutation, Point mutation, Thalassemia