Comparison among plaque assay, tissue culture infectious dose (TCID ) 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 pub- lic health because the rapid change in the SARS-CoV-2 genome can alter viral phenotypes such as virulence, transmissi- bility 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 de- velopment.
 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 infectiousdose (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 de- pending on the SARS-CoV-2 variant, being Mu, the variant reaching a higher viral titer with fewer viral copies.

Rapid Detection of SARS-CoV-2 in Clinical and Environmental Samples via a Resonant Cavity SERS Platform within 20 min

“…The characteristic peaks of the S antigen at 1001, 1230–1300 (amide III), and 1445 cm –1 can be observed at the lowest concentration, which indicates that the LOD for SARS-CoV-2 virions on the VRC substrate is about 3.5 × 10 –4 PFU/mL. According to previous studies, which quantitatively explored the relationship between the titer and viral load for SARS-CoV-2, 1 PFU/mL is about 10 5 –10 6 copies/mL. , Therefore, the LOD of our VRC substrate is about 35–350 copies/mL. The LOD test for the detection of SARS-CoV-2 inactivated vaccine samples was also carried out as shown in Figure S16a,b.…”

“…According to previous studies, which quantitatively explored the relationship between the titer and viral load for SARS-CoV-2, 1 PFU/mL is about 10 5 −10 6 copies/mL. 63,64 Therefore, the LOD of our VRC substrate is about 35−350 copies/mL. The LOD test for the detection of SARS-CoV-2 inactivated vaccine samples was also carried out as shown in Figure S16a,b.…”

High Throughput Determination of Infectious Virus Titers by Kinetic Measurement of infection-Induced Changes in Cell Morphology

“…Our TCID50 assay, which depends on CPE evaluation at a single timepoint, exhibits a CV of about 45 % with a single determination per sample. This is already quite good for such a traditional infectivity assay, where a variability of ±0.5 log10 is not uncommon [6,7]. However, to reach the precision of the KIT assay, data from six measurements, equaling six 96-well plates, would have to be averaged (Figure 7) [8].…”

In vitro and in silico evaluation of antiretrovirals against SARS-CoV-2: A drug repurposing approach

“…After 3 days of incubation at 37 °C, the monolayers were washed with PBS, fixed with 4% formaldehyde and stained with 2% crystal violet solution (Sigma-Aldrich). Plaques were counted and used to calculate the number of plaque-forming units per milliliter (PFU/mL) [36] . The difference in the viral titer after treatment compared to the untreated control was expressed as inhibition percentage.…”