Development and Optimization of a Colorimetric Loop-Mediated Isothermal Amplification (C-LAMP) Assay for Screening of Beta-Thalassemia Mutations

Abstract

To develop and optimize a colorimetric loop-mediated isothermal amplification (C-LAMP) assay for equipment-minimal screening of four common HBB mutations (CD15, CD26/HbE, CD41/42, IVS1-5) prevalent in the Ciayumajakuning population, West Java, Indonesia.
Material and Methods: Allele-specific LAMP primers were designed for each target, with discriminating bases at the 3′ terminus of the FIP/BIP segments; a dual-mismatch strategy was additionally applied for CD26/HbE to widen allele discrimination. Synthetic gBlock templates served as positive controls. Four colorimetric indicators (phenol red, malachite green, propidium iodide, and a commercial fluorescence dye) were compared for visual interpretability, equipment dependency, and cost. Reactions were incubated at 65°C for 60 minutes in a closed-tube format to minimize carryover contamination.
Results: Primer sets for all four targets, plus wild-type, produced specific amplification confirmed by gel electrophoresis, with no signal in wild-type or no-template controls. Phenol red produced the clearest unaided-eye color transition at the lowest cost without auxiliary equipment, outperforming the three comparator indicators, each limited by specificity or equipment dependency. Reproducibility, lyophilized reagent stability, and time-to-color kinetics were not assessed in this developmental phase.
Discussion/Conclusion: The optimized C-LAMP protocol offers a low-cost, instrument-minimal alternative for first-line beta-thalassemia screening, intended to complement centralized confirmatory diagnostics (PCR, RDB, Sanger sequencing) within a tiered screening framework. Reproducibility, lyophilized reagent-stability, and reaction-kinetics studies are planned as follow-up work. Primer sequence details are withheld pending patent application (Universitas Indonesia, Direktorat Inovasi/DJKI).