Fig 1.
SARS-CoV-2 RT-qPCR CDC EUA N1 probe assay.
A. RT-qPCR (CDC EUA N1 probe assay) targeting SARS-CoV-2 Wuhan-Hu-1 N gene in vitro transcribed (IVT) RNA. Log dilution series of N gene IVT RNA ranging from 108 to 10 copies of the N gene underwent reverse transcription. Fluorescence data were collected and processed using a 40 cycle cut-off on an AriaMx Real-Time PCR System. B. Schematic of the pMiniT2.0 SARS-CoV-2 Wuhan-Hu-1 N gene in vitro transcription construct with T7 RNA polymerase promoter driving full-length (1260 nt) N gene RNA production. C. DNase-treated and purified SARS-CoV-2 Wuhan-Hu-1 N gene IVT RNA on a non-denaturing 1% agarose gel stained with ethidium bromide.
Fig 2.
SARS-CoV-2 fluorescent RT-LAMP assay.
A. Fluorescent RT-LAMP assay targeting SARS-CoV-2 Wuhan-Hu-1 N gene IVT (GenBank: MN908947) RNA. Log-dilution series of N gene IVT RNA ranging from 108 to 10 copies of the N gene was added to fluorescent RT-LAMP reactions containing six LAMP primers (FIP/BIP, F3/B3, and LF/LB) and an 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 curve. B. Standard Curve of fluorescent RT-LAMP assay C. Melt curve of the fluorescent RT-LAMP products produced from the reactions in Fig 2A.
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.
Fig 4.
SARS-CoV-2 live virus CDC EUA N1 RT-qPCR assay.
A. RT-qPCR using CDC EUA N1 probe assay targeting SARS-CoV-2 (WA1/2020) and, B. targeting SARS-CoV-2 B.1.1.7 (Alpha) (20I/501Y.V1) in infected Vero cells. Culture supernatants were log-serially ranging from 107 pfu/ml to 10 pfu/ml. RNA was purified from cell culture supernatants and reverse transcribed and cDNA was used in an RT-qPCR reaction in the CDC EUA N1 Probe Assay. Fluorescence data were collected and processed using a 40 cycle cut-off on an AriaMx Real-Time PCR System.
Table 1.
Fluorescent RT- LAMP on SARS-CoV-2 positive NP swab samples.
Table 2.
Fluorescent RT- LAMP of SARS-CoV-2 negative NP swab samples.