Development of Visual Detection of α-Thalassemia-1 (the – –SEA Deletion) Using pH-Sensitive Loop-Mediated Isothermal Amplification

Abstract

Detection of α-thalassemia-1 (α-thal-1) carriers provides valuable insight for genetic consulting in prevention and control programs for couples who are at risk of conceiving a fetus with severe thalassemia, both Hb Bart’s hydrops fetalis and hemolytic Hb H disease. The traditional method is complicated, time-consuming and requires high instrument cost and expertise. Loop-mediated isothermal amplification (LAMP) based on pH-sensitive dye technology, shows all the characteristics required of a real-time analysis with simple operation for potential use in the clinical diagnosis of high incidence α-thal-1 [Southeast Asian (SEA) or – –SEA deletion]. Four primers specific for six distinct regions of the α-globin gene deletion were designed and analyzed by LAMP using the pH-indicator dye, phenol red. The amplification of the – –SEA deletion changed the color of phenol red from pink to orange. The diagnostic ability of detection of the – –SEA deletion by pH-sensitive LAMP was validated using both known and unknown blood samples and compared to the conventional polymerase chain reaction (PCR) method. Color inspection of pH-sensitive LAMP products could clearly identify the – –SEA deletion. There was no cross reaction with a normal α-globin gene, α-thal-1 Thai (– –THAI deletion), α-thal-2 [–α3.7 (rightward) and –α4.2 (leftward) deletion] and β-thalassemia (β-thal). Detection of the SEA deletion by pH-sensitive LAMP was consistent as compared to conventional PCR. The pH-sensitive LAMP method developed for this deletion carrier diagnosis has high sensitivity, specificity, simplicity, and requires simple instrumentation that makes it applicable for resource-limited laboratories in rural areas of developing countries.