Colorimetric LAMP assay enables rapid detection of Hb Lepore and Hb Sicilian deletions in the β-globin gene cluster with minimal instrumentation.

Abstract

Introduction: Accurate and timely detection of hemoglobin (Hb) variants, including Hb Lepore and Hb Sicilian, is crucial for diagnosing and managing β-thalassemia. This study aimed to develop a simple and rapid colorimetric loop-mediated isothermal amplification (LAMP) assay for direct identification of these deletions without the need for complex laboratory equipment.

Methods: Blood samples from patients with confirmed Hb Lepore and Hb Sicilian deletions were analyzed. Genomic DNA was extracted, and LAMP primers targeting deletion breakpoints were designed using PrimerExplorer (Eiken Chemical Co), with the GAPDH PCR module (Bio-Rad Laboratories, Inc) as an internal control. Reactions were performed under isothermal conditions. Colorimetric detection was performed using CYTO24 dye, allowing visual assessment, while real-time fluorescence monitoring was carried out using CYTO 9 dye.

Results: The LAMP assay accurately detected both deletions within 30 minutes, showing concordant results with reference methods in the tested samples. The colorimetric readout allowed easy visual interpretation without electrophoresis or specialized instruments, supporting its use in clinical and research settings.

Discussion: The developed colorimetric LAMP assay provides a simple, fast, and reliable alternative to polymerase chain reaction-based methods for screening hemoglobinopathies involving large deletions. Its visual detection and minimal equipment requirements make it suitable for low-resource or point-of-care settings. Further validation with larger cohorts and additional variants is recommended to enhance the assay's clinical utility.