June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Development of a rapid viability RT-PCR assay for detection of infectious SARS-CoV-2
Author Affiliations & Notes
  • Judith Veugen
    Medical Microbiology, Infectious Diseases & Infection Prevention, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands
    University Eye Clinic Maastricht, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands
  • Tom Schoenmakers
    Clinical Chemistry & Hematology, Zuyderland Medisch Centrum Heerlen, Heerlen, Limburg, Netherlands
  • Inge van Loo
    Medical Microbiology, Infectious Diseases & Infection Prevention, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands
  • Mathie Leers
    Clinical Chemistry & Hematology, Zuyderland Medisch Centrum Heerlen, Heerlen, Limburg, Netherlands
  • Rudy Nuijts
    University Eye Clinic Maastricht, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands
  • Paul Savelkoul
    Medical Microbiology, Infectious Diseases & Infection Prevention, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands
  • Mor Dickman
    University Eye Clinic Maastricht, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands
  • Petra Wolffs
    Medical Microbiology, Infectious Diseases & Infection Prevention, Maastricht Universitair Medisch Centrum+, Maastricht, Limburg, Netherlands
  • Footnotes
    Commercial Relationships   Judith Veugen None; Tom Schoenmakers None; Inge van Loo None; Mathie Leers None; Rudy Nuijts None; Paul Savelkoul None; Mor Dickman None; Petra Wolffs None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 612. doi:
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      Judith Veugen, Tom Schoenmakers, Inge van Loo, Mathie Leers, Rudy Nuijts, Paul Savelkoul, Mor Dickman, Petra Wolffs; Development of a rapid viability RT-PCR assay for detection of infectious SARS-CoV-2. Invest. Ophthalmol. Vis. Sci. 2023;64(8):612.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped virus that causes Coronavirus disease 2019 (COVID-19), which has rapidly become a global health problem. The most commonly used methods to detect SARS-CoV-2 RNA cannot conclude whether samples that tested positive for SARS-CoV-2 contain intact and therefore infectious virus particles. Currently, cell culture is the gold standard to assess viability of SARS-CoV-2. However, this time-consuming technique requires a biosafety level 3-laboratory, making it impractical in most clinical settings. The aim of this study was to develop a rapid and sensitive viability PCR assay that can distinguish between infectious and non-infectious SARS-CoV-2 in one day. In viability PCR, samples are pre-treated with a photoreactive dye that binds to DNA of non-infectious virus and thus prevents DNA amplification.

Methods : PMAXX was used as a photoreactive dye. Mixtures with concentrations of 100%, 50%, 10%, 1%, 0.1%, and 0% viable SARS-CoV-2 were prepared, and each concentration was divided in a PMAXX-treated group and a non-PMAXX-treated group. Reverse transcription-polymerase chain reaction (RT-PCR) using an in-house developed viability assay was applied to detect viral infectivity of SARS-CoV-2. The smaller the delta Ct value, the higher the risk of infection from SARS-CoV-2. Each experiment was performed in duplicate for both high, cycle threshold (Ct) value 16, and low, Ct value 30, viral loads.

Results : In samples with a high viral load mixtures containing 100%, 50%, 10%, 1%, 0.1%, and 0% viable SARS-CoV-2 showed mean delta Ct values of 2.1, 3.5, 6.2, 9.4, 12.3, and 14.2 respectively.
In samples with a low viral load mixtures containing 100%, 50%, 10%, and 1% viable SARS-CoV-2 showed mean delta Ct values of 2.4, 3.8, 5.8, and 8.5 respectively. For the mixtures containing 0.1% and 0% viable SARS-CoV-2 the delta Ct values were ≥11.6 and ≥11.5 respectively.

Conclusions : High and low viral load mixtures containing different percentages of viable SARS-CoV-2 showed similar results, making this assay suitable for both high and low viral loads. In the current study, a rapid viability RT-PCR assay has been developed that can distinguish between infectious and non-infectious SARS-CoV-2 based on the delta Ct values between the PMA-treated and PMA-untreated groups.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

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