Lyophilised colourimetric LAMP for visual readout with dual colour indicators

Abstract

Loop-mediated isothermal amplification (LAMP) is increasingly recognised as a practical alternative to PCR for pathogen detection, offering rapid turnaround time, a constant operating temperature, and compatibility with a wide range of detection methods. Colourimetric LAMP has gained popularity due to its potential for instrument-free readout, making it suitable for molecular diagnostics in low-resource settings. Despite these advantages, its adoption at the point-of-care remains limited as it has been widely used in liquid format and therefore restricted to the availability of cold-chain storage and trained personnel. This work introduces the development and optimisation of ready-to-use lyophilised colourimetric LAMP (lyo-cLAMP) that does not require cold-chain, additional reagents, or manual intervention beyond the addition of extracted nucleic acids from a sample, which indicates the presence of a target of interest by colour change after amplification. A variety of pH and metal indicators were screened and combined to evaluate their synergy, identifying four combinations with high discrimination between positive and negative amplification. The performance of lyo-cLAMP was assessed with synthetic SARS-CoV-2 RNA, comparing it to the liquid format and achieving the same limit-of-detection. Lastly, the translation of lyo-cLAMP to diagnostic applications was demonstrated by screening positive SARS-CoV-2 residual clinical samples, achieving high accuracy. The developed lyo-cLAMP is compatible with any LAMP assay, allowing for rapid adaptation to new targets, which is particularly valuable in outbreak scenarios.