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.