Rapid molecular diagnostics of large deletional β0-thalassemia (3.5 kb and 45 kb) using colorimetric LAMP in various thalassemia genotypes

Abstract

Background: β-thalassemia is an inherited disorder that is reported worldwide. Two common β0-thalassemia mutations (3.5 kb and 45 kb deletions) are prevalent in Southeast Asia and Thailand. Identification of these defects is essential to population screening and prenatal diagnosis. We aimed to develop colorimetric LAMP based on a phenol red indicator and validate it on various thalassemia genotypes. Method: Colorimetric LAMP assays for detecting β0-thalassemia 3.5- and 45-kb deletions were developed and validated on 254 routine clinical samples. The results of the assays could be interpreted by the naked eye and compared with the gold standard gap-PCR. Results: A total of 254 samples related to seven phenotypes and 27 different genotype groups showed 100% concordance between the colorimetric LAMP assays and gap-PCR for detecting β0-thalassemia (3.5- and 45-kb deletions). The sensitivity, specificity, NPV, and PPV were calculated as 100% for both β0-thalassemia 3.5- and 45-kb deletion detection. The comparison of the usefulness of colorimetric LAMP assays and conventional methods was demonstrated in this study. Conclusions: The developed colorimetric LAMP assays are rapid, simple, and highly cost effective and can be interpreted by the naked eye. These assays should be applied for screening deletional β0-thalassemia in routine settings or small community hospitals in remote areas where thalassemia is highly heterogeneous.

Keywords

• 3.5 kb deletion
• 45 kb deletion
• β-thalassemia
• Loop-mediated isothermal amplification
• Molecular testing