Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications

  1. Nicky Craig
  2. Sarah L. Fletcher
  3. Alison Daniels
  4. Caitlin Newman
  5. Marie O’Shea
  6. Amanda Warr
  7. Christine Tait-Burkard

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

An enormous global effort is being made to study SARS-CoV-2 and develop safe and effective treatments. Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify host factors and treatments to combat the infection. However, quantification of released virus often requires lengthy procedures, such as endpoint dilution assays or reinfection with engineered reporter viruses. Quantification of viral RNA in cell supernatant is faster and can be performed on clinical isolates. However, viral RNA purification is expensive in time and resources and often unsuitable for high-throughput screening. Here, we show a direct lysis RT-qPCR method allowing sensitive, accurate, fast, and cheap quantification of SARS-CoV-2 in culture supernatant. During lysis, the virus is completely inactivated, allowing further processing in low containment areas. This protocol facilitates a wide array of high- and low-throughput applications from basic quantification to studying the biology of SARS-CoV-2 and to identify novel antiviral treatments in vitro.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2021.11.30.470550: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: amplicon set V3, and validated against the patient isolate sequence.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Resource availability: Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Christine Tait-Burkard (Christine.burkard(at)roslin.ed.ac.uk) Experimental model and subject details: Cells and viruses: Vero E6 (ATCC CRL-1586) cells were maintained as monolayer cultures in Dublecco’s modified Eagle’s medium (DMEM, Sigma), supplemented with 10% heat inactivated Fetal Bovine Serum (FBS, (Gibco), 1X Ultraglutamine-I (Lonza)
    Vero E6
    suggested: None
    Recombinant DNA
    SentencesResources
    (Supplementary Table S1), containing parts of N (orf10) were cloned into the pCR-Blunt-II vector using the PCR Blunt Topo kit according to the manufacturer’s instructions and transformed into 10-beta CaCl2-chemically competent bacteria (originally NEB, generated in-house).
    pCR-Blunt-II
    suggested: RRID:Addgene_86688)
    Software and Algorithms
    SentencesResources
    Virus sequence was confirmed by Nanopore sequencing according to the ARCTIC network protocol (https://artic.network/ncov-2019),
    ARCTIC
    suggested: (ARCTIC, RRID:SCR_005989)
    A semilog fit was performed using Graphpad Prism to determine the slope to determine the RT-qPCR efficiency.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2021.11.30.470550v1 on bioRxiv