Ose db 02

Real-time RT-RPA detection was performed using an RT-RPA custom kit (Amp-Future, China) in a 10 μL total reaction volume. The RPA reaction mixture consisted of 5.9 μL of buffer A (rehydration buffer), 0.4 μL of each primer (10 μM), 0.12 μL of probe (10 μM), 1.68 μL of nuclease-free water, and 1 μL of RNA template. The total mixture was added to the test tube containing lyophilized enzyme powder and then carefully dropped 0.5 μL of buffer B (280 mM magnesium acetate) into the inner wall of each reaction tube. After capping, forcefully invert the tube 5–8 times to mix the contents, then centrifuge. We added a mixing step after the reaction started to boost reaction efficiency. Tubes were incubated for 5 min (2 min for reactions containing 10³ or more template copies) in a thermostatic metal dry bath (OSE-DB-02, Tiangen Biotech Co. Ltd., China), then briefly mixed and centrifuged before being transferred to a fluorescence detector (Cobas z 480, Roche, Switzerland), and measured at 42°C for 15 min.