Comparison among plaque assay, tissue culture infectious dose (TCID50) and real-time RT-PCR for SARS-CoV-2 variants quantification

Abstract

Background and objectives: SARS-CoV-2 variants of concern (VOC) and interest (VOI) pose a significant threat to public health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissibility and the ability to evade the host response. Hence, SARS-CoV-2 quantification techniques are essential for timely diagnosis and follow-up. Besides, they are vital to understanding viral pathogenesis, antiviral evaluation, and vaccine development.

Materials and methods: Five isolates of SARS-CoV-2: D614G strain (B.1), three VOC (Alpha, Gamma and Delta), and one VOI (Mu) were used to compare three techniques for viral quantification, plaque assay, median tissue culture infectious dose (TCID₅₀) and real-time RT-PCR.

Results: Plaque assay showed viral titers between 0.15 ± 0.01×10⁷ and 1.95 ± 0.09×10⁷ PFU/mL while viral titer by TCID₅₀ assay was between 0.71 ± 0.01×10⁶ to 4.94 ± 0.80×10⁶ TCID₅₀/mL for the five SARS-CoV-2 isolates. The PFU/mL titer obtained by plaque and the calculated from TCID₅₀ assays differed by 0.61 log10, 0.59 log10, 0.59 log10 and 0.96 log10 for Alfa, Gamma, Delta, and Mu variants (p≤0.0007), respectively. No differences were observed for the D614G strain. Real-time PCR assay exhibited titers ranging from 0.39 ± 0.001×10⁸ to 3.38 ± 0.04×10⁸ RNA copies/μL for all variants. The relation between PFU/mL and RNA copies/mL was 1:29800 for D614G strain, 1:11700 for Alpha, 1:8930 for Gamma, 1:12500 for Delta, and 1:2950 for Mu.

Conclusion: TCID₅₀ assay was comparable to plaque assay for D614G but not for others SARS-CoV-2 variants. Our data demonstrated a correlation among PFU/mL and E gene RNA copies/μL, units of measure commonly used to quantify the viral load in diagnostic and research fields. The results suggest that the proportion of infectious virions in vitro changes depending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.

Keywords: Median tissue culture infectious dose assay; Plaque assay; Real-time reverse transcription-polymerase chain reaction; SARS-CoV-2 variants; Virus titer.