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 [–α^3.7 (rightward) and –α^4.2 (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.

Keywords:

• α-Thalassemia-1 (α-thal-1)
• molecular diagnosis
• loop-mediated isothermal amplification
• prenatal diagnosis
• naked eye detection

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

The authors gratefully acknowledge the financial support provided by the Thammasat University Research Fund under the Thammasat University Research Scholarship [Contract No. 035/2557].