RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

Competing interests' statement S-MF has received funding from Gilead, Bayer AG, Merck, Black Belt Therapeutics and Alesta Therapeutics, has consulted for Fund+ and is in the advisory board of Alesta Therapeutics. TGPG has consulted for Boehringer Ingelheim. All other authors declare no competing interests. Resource AvailabilityThis study did not generate new unique reagents, except for genetically manipulated cell lines based on commercially available constructs. Reagents generated in this study will be made available on request through the lead author or the collaboration partner that generated the resource, but we may require payment and/or a completed Materials Transfer Agreement if there is potential for commercial application. Further requests for resources should be directed to the lead contact, Sarah-Maria Fendt

RT-LAMP detection of SARS-CoV-2 has been shown as a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validated the optimized RT-LAMP assay for the detection of SARS-CoV-2 in raw nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings with limited economic resources.