Single-tube multiplex real-time PCR with EvaGreen and high-resolution melting analysis for diagnosis of α(0)-thalassemia--(SEA),--(THAI), and--(CR) type deletions

Regions with a high prevalence of α-thalassemia (α-thal) require simple, rapid, and accurate tests for carrier screening and prenatal diagnosis. Diagnosis of multiple deletions in a single tube is necessary to clearly identify individuals with α(0)-thalassemia in the routine setting, especially in at-risk couples. Therefore, we aimed to develop a single-tube multiplex real-time PCR with EvaGreen and high-resolution melting (HRM) analysis for the identification of α(0)-thalassemia Southeast Asian (SEA), Thai and Chiang Rai (CR) type deletions. The results of the HRM analysis indicated that the amplified fragments from α(0)-thal--(CR),--(THAI),--(SEA), and the wild-type α-globin gene had specific peak heights at mean melting temperature (T(m)) values of 85.40°C, 86.50°C, 87.65°C, and 91.04°C, respectively. The frequencies of α(0)-thal--(SEA),--(THAI),--(CR) obtained from routine testing in 2,135 samples were 17.89%, 0.19% and 0.19%, respectively. This method would be useful for preventing Hb Bart’s hydrops fetalis. Detection of multiple deletions in a single run is cost-effective, highly accurate and timesaving. This technique could enable wider α-thalassemia diagnosis in high prevalence areas and served as an example for thalassemia routine setting.