SARS-CoV-2
Viral RNA used in this study was provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at UTMB. SARS-CoV-2 strain USA_WA1/2020 was provided at a reported concentration of 6x104 PFU/µl or approximately 6x107 genomic equivalents/µl of extracted RNA.
Patient Samples
De-identified samples that were suspected to contain SARS-CoV-2 genomic material were collected at UTMB for further laboratory analysis under UTMB IRB protocol #95-111. Briefly, nasopharyngeal swabs were collected and placed in transport media per clinic protocol for COVID screening. The transport media potentially containing active virus was heat inactivated at 95°C for 65 minutes by the UTMB clinical laboratories prior to processing for RNA extraction and cDNA synthesis as described as below.
Human sample Ethics statement
All data acquisition and experimental procedures were performed in accordance with relevant guidelines and regulations. All clinical samples were obtained as de-identified patient samples and considered medical waste under protocol #95-111. This protocol was approved by the Institutional Review Board (IRB) of the University of Texas Medical Branch at Galveston, TX. All procedures performed within have been approved by the Institutional Biosafety Committee (IBC) under Notification of Use (NOU) #2020113.
Viral RNA isolation and cDNA synthesis
The virus was inactivated using Trizol followed by modified chloroform separation and RNA isolation using the Qiagen RNeasy Mini Kit (Qiagen). The cDNA synthesis was carried out using the iScript Select cDNA Synthesis Kit (Bio-Rad) following the manufacturer’s protocol. For cDNA synthesis we used 10 µL of RNA and 3 µL of random primers (Bio-Rad) as per the manufacturer instructions.
Quantitative reverse transcriptase Real time PCR for SARS-CoV-2
For detection of clinical samples and quantification viral RNA (provided by WRECEVA) we used the CDC’s nucleocapsid (N1, N2, and N3 primers as described) rRT-PCR for COVID-19 (10, 11). To determine the limit of detection by RPA-LF, we performed 10-fold serial dilutions of the N gene plasmid provided in the kit and compared it to serially diluted cDNA transcribed from SARS-CoV-2 RNA.
SARS-CoV-2 primer and probe design and position:
Primers were selected from the commercially available CDC real-time Rt PCR for detection 2019-Novel Coronavirus (10, 11). The assay consists of three primer pairs targeting the nucleocapsid gene (Fig 1A). We used the primers in multiple combinations at 40°C (Fig 1B) to determine the pair that would work the best under isothermal conditions. Once we identified which primer pair functioned more efficiently under these conditions (N1FW and N3RV), several probes were designed based on the assay’s existing probes. The modifications of the RPA-LF probes required at least 50 base pairs and include FAM (5’-carboxy fluorescein amidite) at the 5’ end, an internal dSpacer that acts as an exonuclease cut-site, and a C3-Spacer at the 3’ end, as suggested by the manufacturer (TwistDx, UK). Results are shown for the CDC N1 probe (2019-nCoV_N1-P) (IDT-Petaluma, CA) modified to meet the above criteria (Fig 1A). All primers and probe were BLASTed (NCBI GenBank) to ensure no cross-reactivity with other organisms, only SARS-CoV-2 sequences returned. To enable for detection by lateral flow, the reverse primer was biotinylated at the 5’ end. Table 1 shows the primers and probe sequences used in the RPA-LF assay.
RPA reaction and LF reading
The amplification mixture was comprised of: 1) forward primer (4.8µL, 5µM), 2) biotinylated reverse primer (4.8µL, 5µM), 3) FAM-labeled probe (0.6µL, 5μM), 4) magnesium acetate (2.5µL, 288mM), 5) 6.6 µL of water), and 6) the rehydrated cocktail (Twist amp nfo RPA kit -TwistDx, UK). Viral cDNA or control plasmid (2.5 μL) was immediately added to the mixture and subjected to amplification at 42.0°C for 40 minutes using a dry bath incubator (VWR). The RPA product was diluted 1:50 in 100 μL of dipstick assay buffer in a 1.5 μL Eppendorf tube. The bottom tip of the lateral flow strip (Ustar Biotechnologies, Hangzhou) was then immersed in the sample making the amplification product run upwards by capillarity. Viral cDNA amplification was confirmed with the naked eye after 5 minutes by the appearance of the test band in the lower part of the strip in addition to the control band.
Statistical analysis
Probit analysis was conducted for the lowest concentrations detected of N gene plasmid and viral genome cDNA samples. Statistical analysis was done using free software from https://astatsa.com/Logit_Probit or XLSTAT.