Enhancing Rapid Detection of COVID-19: Optimization of RT-LAMP Assays for Improved Sensitivity and Specificity

Background: The COVID-19 pandemic has underscored the critical need for rapid and reliable diagnostic methods to detect the SARS-CoV-2 virus and effectively curb its transmission. Traditional diagnostic methods, such as RT-PCR, although accurate, often require extensive laboratory infrastructure and time, which can hinder timely public health interventions. Objectives: This study aimed to optimize a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the direct detection of viral RNA from nasopharyngeal swabs, eliminating the need for nucleic acid extraction and reducing reliance on sophisticated laboratory equipment. Methods: The optimization process involved several key steps, including adjustments in sample preparation techniques, fine-tuning the concentration of proteinase K, and incorporating guanidine hydrochloride and betaine to enhance the assay's sensitivity. Results: The optimized RT-LAMP assay achieved a sensitivity of approximately 85.25% and a specificity of 100% when utilizing extracted RNA. In comparison, the direct use of pharyngeal swab samples yielded a sensitivity of 83.61% and a specificity of 86.67%. The assay successfully identified viral RNA with cycle threshold (CT) values up to 35, indicating its capability to detect low viral loads comparable to those identified by standard RT-PCR methods. Test positivity cut-offs were based on a colorimetric shift and confirmed by gel electrophoresis. Conclusions: This RT-LAMP assay presents a promising alternative to RT-PCR for rapid COVID-19 diagnostics, particularly in resource-limited settings. By providing a balance of efficiency, accuracy, and accessibility, it can facilitate timely public health responses and enhance the capacity for widespread testing during pandemics.