RT-LAMP assay
Assays were assembled in total reaction volumes of either 12.5 μl (for LAMP assays using isolated RNA) or 20 μl (for swab–to–RT-LAMP assays). Master mixes were prepared at room temperature for each reaction immediately before use with either 6.25 or 10 μl, respectively, of the WarmStart Colorimetric RT-LAMP 2X Master Mix (M1800, New England Biolabs) and 1.25 or 2 μl, respectively, of the 10× primer mix, filled up to 11.5 or 19 μl with nuclease-free water (AM9937, Ambion). Values given are for one reaction: For a 96-well plate, 100 times larger volumes were used, and the LAMP mix was distributed to the wells of a 96-well plate (4ti-0960/C, Brooks Life Sciences or 0030128672, Eppendorf) before pipetting 1 μl of sample into each well of the plate; for details, see previous paragraph. Plates were prepared immediately before use to limit exposure of the LAMP reagents to atmospheric CO2 (to prevent acidification of the reaction) and kept on an ice-cold metal block. Plates were sealed using a transparent adhesive foil (GK480-OS, Kisker Biotech), and the reactions were incubated in a PCR cycler at 65°C for 15 to 60 min with the lid heated to 75°C. To perform measurements at the indicated time points, the reactions were taken out of the PCR cycler and placed into an ice cold metal block for 30 s. This intensifies the color before the measurement. Photographs were taken with cell phone cameras or the scanner function of an office copying machine.