Development and validation of loop-mediated isothermal amplification methods for rapid and sensitive nucleic acid detection in clinical samples

Abstract

What if diagnostic laboratories could detect infectious pathogens in under an hour without expensive thermocyclers? Loop-mediated isothermal amplification (LAMP) offers this possibility through its unique isothermal reaction mechanism. This research describes the development and comprehensive validation of LAMP-based methods for detecting nucleic acids in clinical specimens. The assay employed six specifically designed primers targeting conserved genomic regions, enabling high-fidelity amplification at a constant temperature of 63 °C using Bst DNA polymerase. Clinical validation encompassed 847 samples collected between March 2023 and November 2024 from the molecular diagnostics laboratory at Lyon Institute of Biomedical Sciences. The optimized LAMP protocol achieved a diagnostic sensitivity of 96.7% and specificity of 98.2% when compared against real-time quantitative PCR as the reference standard. The limit of detection reached 12.4 copies per microliter, with complete amplification occurring within 47.3 minutes on average. Reproducibility testing demonstrated coefficient of variation values below 8.3% across different operators and equipment batches. Cross-reactivity panels confirmed no false-positive reactions with phylogenetically related organisms. The colorimetric detection system using hydroxynaphthol blue enabled visual result interpretation without specialized instrumentation. Cost analysis revealed per-test expenses of approximately €8.50, representing a 44% reduction compared to conventional PCR methods. These findings establish LAMP as a viable alternative for resource-limited settings and point-of-care applications where rapid turnaround and minimal infrastructure requirements are essential considerations for clinical implementation.