RT-LAMP assay
RT-LAMP was performed with Isothermal Master mix reagent ISO-004 (Canon medical systems) using the Genelyzer FIII real-time fluorescence detection platform (Canon medical systems). The reaction mixture (total volume = 25 μL) contained 15 μL of Isothermal Master Mix, 1 U of AMV Reverse Transcriptase (Nippon gene), 20 pmol (each) of FIP and BIP primers, 5 pmol (each) of F3 and B3 outer primers, 10 pmol (each) of F and B loop primers, and 5 μL of RNA sample (template). The reaction was performed at the following conditions: for the ORF1b-1 primer set, 68°C for 20 min, followed by a dissociation analysis at 95°C–75°C with the temperature change rate of 0.1°C/s; for the ORF1b-2 primer set, 67°C for 20 min, followed by a similar dissociation analysis. Extracted RNA from samples or heat inactivated swab samples were used as a template. Synthesized RNAs containing the target sequence of the LAMP assay were used as positive controls. Nonspecific amplification was excluded by comparing the melting temperature to that of the positive control [13].