Naked eye detection of α⁰ thalassemia--^sea type using hydroxynaphthol blue loop-mediated isothermal amplification

Abstract

© 2020 Somphon Phraephan, Suphatra Watanasatitarpa, Sorasak Intorasoot. BACKGROUND: Alpha-thalassemia is a major health problem for worldwide populations due to its recent large-scale global movements to many other parts of the world. The most effective method to prevent the spread of the worst form of α-thalassemia disorder, hemoglobin Bart’s hydrops fetalis, is to identify the genotypes of an α0 thalassemia carrier and then conduct subsequent genetic counseling. AIM: This research was aimed to develop a new method for the detection of α0 thalassemia (Southeast Asia or SEA)--SEA type using hydroxynaphthol blue loop-mediated isothermal amplification (HNB-LAMP) and by visually reading results using the naked eye. METHODS: This experimental research was conducted at Rangsit University, Pathum Thani Provinces, Thailand, from 2016 to 2018. Two sets of LAMP primers were used, one set of primer was used to detect the two existing α genes and the other set used in the detection of two α genes deletion--SEA types which were designed based on the nucleotide sequence of two α genes deletion from four cases of the α0 thalassemia carrier--SEA type. HNB-LAMP was developed, and then, 77 cases from subjects were evaluated by comparing them with a conventional polymerase chain reaction (PCR). RESULTS: The newly developed HNB-LAMP shows a high diagnostic sensitivity and diagnostic specificity, 100% in correct interpretation, from 10 cases of α0 thalassemia carriers--SEA type, 29 cases of α+ thalassemia carrier-α3.7 type, 8 cases of hemoglobin constant spring/hemoglobin Pakse′ (HbCS/HbPS), and 30 standard cases. CONCLUSION: This newly developed HNB-LAMP is suitable for in the field applications for detecting the presence of α0 thalassemia--SEA type due to its high diagnostic sensitivity, high diagnostic specificity, and, most notably, the ability to be detected with the naked eye.