Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification

Laura E Lamb; Sarah N Bartolone; Elijah Ward; Michael B Chancellor

To establish the optimal conditions for RT-LAMP using a COVID-19 PCR-standard designed from nucleotide 2941-3420 of the COVID-19 Wuhan-Hu-1 complete genome (MN908947), several primer sets, ranges of temperatures (57-65°C), and incubation times (15-45 mins) were tested. The best amplification results were obtained at 63 ° C for 30 minutes as indicated by a banding pattern after electrophoresis on a gel (Fig 1) . Positive reactions containing SYBR Green I could be observed by naked eye by a color change from orange to yellow (Fig 1-top 1) . In order to determine the lower detection limit of the RT-LAMP reaction for COVID-19, a dilution series ranging from 0.204 fg to 10 ng COVID-19 was amplified (Fig 2) . The limit of detection was approximately equivalent to 1.02 fg. This dilution series was run in parallel with qRT-PCR using primers that targeted this same region of COVID-19 genome; the qRT-PCR Ct values for the dilution series are reported in Supplemental Table 1 .