Utilizing loop-mediated isothermal amplification (LAMP) for detecting hemoglobin Constant Spring and hemoglobin Pakse mutations amidst the high prevalence and genetic heterogeneity of thalassemia in Thailand

Abstract

Thalassemia is a genetic disorder with significant prevalence in Southeast Asia, particularly in Thailand, where hemoglobin (Hb) Constant Spring (Hb CS) and hemoglobin Pakse (Hb PS) mutations are common. These mutations, resulting from stop codon alterations in the 2-globin gene, can lead to severe phenotypes such as non-deletional Hb H disease. This study aimed to develop and evaluate a novel colorimetric loop-mediated isothermal amplification (LAMP) assay for detecting Hb CS and Hb PS mutations. A total of 282 samples with several genotypes were recruited in the study. We developed LAMP assay, using a phenol red pH indicator, which provided visual detection of DNA amplification within 35 minutes at 65-C. Both assays demonstrated a lower limit of detection of 0.625 ng/reaction and achieved 100% sensitivity and specificity across 282 DNA samples, validated against standard allelespecific polymerase chain reaction (PCR). Additionally, the assay's minimal equipment requirements and cost-effectiveness make it suitable for use in community hospitals and large-scale screenings. The LAMP assay offers a rapid, accurate, and affordable alternative for Hb CS and Hb PS detection, addressing the challenges of managing thalassemia in genetically diverse and resource-limited regions like Thailand.