A facile Q-RT-PCR assay for monitoring SARS-CoV-2 growth in cell culture

Abstract

19Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the 20 ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to 21 spread around the globe causing immense respiratory disease and mortality. Assays to monitor 22 SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering 23 progress in basic research and drug development efforts. Here we developed a facile Q-RT- 24PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication 25 kinetics from a small amount of cell culture supernatants. Using this assay, we screened the 26 activities of a number of entry, SARS-CoV-2-and HIV-1-specific inhibitors in a proof of concept 27 study. In line with previous studies which has shown that processing of the viral Spike protein by 28 cellular proteases and endosomal fusion are required for entry, we found that E64D and 29 apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with 30 minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly 31 decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an 32 alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease 33 inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 34 replication, albeit the IC 50 values were much higher than that required for HIV-1. Taken together, 35this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-36 throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad 37 number of RNA and DNA viruses. 38 39 40 41 Importance 42 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the 43 COVID-19 pandemic, has quickly become a major global health problem causing immense 44 respiratory disease and social and economic disruptions. Conventional assays that monitor 45 SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction 46 procedures, hampering progress in basic SARS-CoV-2 research and development of effective 47 therapeutics. Here we developed a facile Q-RT-PCR assay to monitor SARS-CoV-2 growth in 48 cell culture supernatants that does not necessitate RNA extraction, and is as accurate and 49 sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA 50 and remdesivir can substantially impede SARS-Cov-2 replication providing novel insight into 51 viral entry and replication mechanisms. This facile approach will undoubtedly expedite basic 52 SARS-CoV-2 research, be amenable to screening platforms to identify therapeutics against 53 SARS-CoV-2 and can be adapted to numerous other RNA and DNA viruses.54 55 56 57 58 59 60 61 62 Observation 63 Severe acute respiratory syndrome coronavirus, SARS-CoV-2, the causative agent of the 64 ongoing COVID-19 pandemic, is continuing to cause substantial morbidity an...

Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection

“…While these open-source approaches may not match the exquisite sensitivity of expensive commercial kits, testing centers must consider whether some reduction in sensitivity is worth increased availability of tests in the face of economic and logistical constraints. Continued refinement of open-source diagnostic methods and their adoption by "pop-up" testing centers [26,46,47] could facilitate expanded testing, both in the current pandemic and in response to novel viruses in the future.…”

“…Validating BEARmix for clinical diagnostics would of course require more extensive side-by-side comparison of BEARmix and a commercial master mix in an actual testing center, and it is likely that the relative performance of BEARmix and other master mixes may differ depending on the primer set used [13]. Additionally, it would be interesting to evaluate BEARmix in combination with direct-addition protocols for saliva testing [46,47]. This basic master mix recipe could be improved in various ways, for instance, by including dUTP and UDG to prevent amplicon contamination, optimizing the conditions for hot-start Taq preparation and reactivation, or testing other public-domain DNA polymerase and reverse transcriptase variants [48].…”

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

“…Extracted RNA was either subjected to Q-RT-PCR analysis for viral RNAs, cellular RNA, or RNA-seq. Viral RNA in cell culture supernatants was quantitated as detailed before (Shema Mugisha et al, 2020b). In brief, 5 μL of supernatant was mixed with 5 μL of 2x lysis buffer (2% Triton X-100, 50mM KCl, 100mM TrisHCl pH7.4, 40% glycerol supplemented with 400u/mL of SuperaseIN (Life Technologies)), followed by addition of 90 μL of 1X core buffer (5 mM (NH4)2SO4, 20 mM KCl and 20 mM Tris–HCl pH 8.3).…”

“…10 μL of this sample was used in a TaqMan-based Q-RT-PCR assay using TaqMan™ RNA-to-CT™ 1-Step Kit (Applied Biosystems, #4392938), alongside with RNA standards, targeting SARS-CoV-2 N gene. The primers and probe sequences are as described before (Shema Mugisha et al, 2020b). To study the interferon (IFN) response, cellular RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) followed by Q-RT-PCR analysis using PowerUp SYBR Green Master Mix (Applied Biosystems).…”

Repurposing Approved Drugs for Guiding COVID-19 Prophylaxis: A Systematic Review

“…Generally, HIV protease inhibitors are used in combination with at least two other anti-HIV drugs (Sadler et al, 2020). Recent in vitro studies demonstrated that amprenavir exhibited a considerable degree of inhibition against SARS-CoV-2 (Mugisha et al, 2020).…”