RT-LAMP Assay for Rapid RNA Detection

The RT-LAMP reaction was prepared using the Loopamp RNA Amplification Kit (Eiken Chemical Co. Ltd., Japan). Each RT-LAMP reaction (25 μl) was added with the inner primers (20 pmol each), outer primers (2.5 pmol each), loop primer (20 pmol each), Fluorescent Detection Reagent (Eiken Chemical Co. Ltd., Japan; 1 μl), and the eluted RNA (5 μl). The RT-LAMP were performed using LA-500 Loopamp real-time turbidimeter (Eiken Chemical Co. Ltd., Japan) according to the following conditions: 90 min at 63 °C followed by 5 min of assay inactivation at 80 °C. The turbidity of RT-LAMP reaction was measured at 650 nm every 6 s. The threshold time (Tt) value was recorded when the turbidity crossed the threshold cut-off value at 0.07 absorbance units [48 (link), 49 (link)].