Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs
Fig 3
SARS-CoV-2 live virus fluorescent RT-LAMP assay.
A. Fluorescent RT-LAMP assay targeting SARS-CoV-2 (USA-WA1/2020 GenBank: MN985325.1), and B. Fluorescent RT-LAMP assay targeting SARS-CoV-2 variant B1.1.7 variant (20I/501Y.V1) in infected Vero cell culture. Culture supernatants were log-serially diluted from 107 pfu/ml to 10 pfu/ml. Fluorescent RT-LAMP was performed using six LAMP primers (FIP/BIP, F3/B3, and LF/LB), and SYBR-like fluorescent dye. Reactions were performed on an AriaMx Real-Time PCR System at 65°C for 30 min with fluorescence monitored in the FAM/SYBR channel. Fluorescence values were plotted against the elapsed reaction time (min) to generate the amplification curves. Virus-infected Vero cell culture supernatants below 102 pfu/ml were not detected by 30 min. C. No Template Control (NTC), and heterologous human coronavirus strains at 106 pfu/ml, OC43 (GenBank: AY585228.1), 229E (GenBank: AF304460.1), and NL63 (GenBank: AY567487.2) in infected Vero cells failed to produce fluorescence signal over the background in 30 min in the fluorescence RT-LAMP assay.