A lyophilized open-source RT-LAMP assay for molecular diagnostics in resource-limited settings

Abstract

A critical bottleneck for equitable access to population-scale molecular diagnostics is the limited availability of rapid, inex pensive point-of-care tests, especially in low- and middle- income countries. Here, we developed an open-source reverse transcription loop–mediated isothermal amplification (RT-LAMP) molecular assay for pathogen detection. It is based on non proprietary enzymes, namely, HIV-1 reverse transcriptase, Bst LF DNA polymerase, and UDG BMTU thermolabile uracil-DNA gly cosylase. Formulated as liquid or lyophilized reaction mixtures, these reagents enable sensitive colorimetric detection of re spiratory samples without the need for prior nucleic acid iso lation. We evaluated our lyophilized RT-LAMP assay on clinical samples with suspected COVID-19 infection, demonstrating high sensitivity and 100% specificity compared with the gold- standard RT–qPCR. Reaction performance was unaffected by prolonged storage of lyophilized reagents at ambient or elevated temperatures. As a proof of concept, we evaluated the robust ness and ease of use of lyophilized RT-LAMP reaction mixes through independent laboratory testing of COVID-19 samples in Ghana. Overall, our open-source RT-LAMP assay provides a f lexible and scalable point-of-care test that can be adapted for rapid detection of various pathogens in resource-limited settings.