Correlating qRT-PCR, dPCR and Viral Titration for the Identification and Quantification of SARS-CoV-2: A New Approach for Infection Management

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in Wuhan, China, in late 2019 and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) represents the gold standard for diagnostic assays even if it cannot precisely quantify viral RNA copies. Thus, we decided to compare qRT-PCR with digital polymerase chain reaction (dPCR), which is able to give an accurate number of RNA copies that can be found in a specimen. However, the aforementioned methods are not capable to discriminate if the detected RNA is infectious or not. For this purpose, it is necessary to perform an endpoint titration on cell cultures, which is largely used in the research field and provides a tissue culture infecting dose per mL (TCID50/mL) value. Both research and diagnostics call for a model that allows the comparison between the results obtained employing different analytical methods. The aim of this study is to define a comparison among two qRT-PCR protocols (one with preliminary RNA extraction and purification and an extraction-free qRT-PCR), a dPCR and a titration on cell cultures. The resulting correlations yield a faithful estimation of the total number of RNA copies and of the infectious viral burden from a Ct value obtained with diagnostic routine tests. All these estimations take into consideration methodological errors linked to the qRT-PCR, dPCR and titration assays.

Keywords: COVID-19; Ct; RNA copies; SARS-CoV-2; TCID50/mL; dPCR; qRT-PCR; viral titration.

Figure 1

Ct values obtained with preliminary RNA extraction (Nextractor shown in green line) and the extraction-free protocol (Allplex severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Extraction-Free, shown in blue line). Ct values are presented as the mean of six replicates for each dilution ± standard error (SE).

Figure 2

Correlation between Ct values obtained with preliminary RNA extraction (Nextractor) and the extraction-free protocol (Allplex SARS-CoV-2 Extraction-Free). Ct values are presented as the mean of six replicates for each dilution ± SE.

Figure 3

Tissue culture infecting dose per mL (TCID50/mL) values obtained by titration on Vero E6 cell cultures of scalar dilutions of the viral stock. TCID50/mL values are presented as the mean of the two different methods (Reed and Muench and Karber) of two replicates for each dilution ± SE.

Figure 4

Correlation between the results obtained from the same set of samples by digital polymerase chain reaction (dPCR, number of RNA copies per µL on a log 10 scale) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) with preliminary RNA extraction (Nextractor Ct values).

Figure 5

Correlation between TCID50/mL results (on a log10 scale) from titration on Vero E6 cells with the Reed and Muench method and Ct values derived from qRT-PCR with preliminary RNA extraction (Nextractor). Ct values are presented as the mean of six replicates for each dilution ± SE, whereas TCID50/mL values are presented as the mean of the two different methods (Reed and Muench and Karber) of two replicates for each dilution ± SE.

Figure 6

The workflow explaining the procedures and protocols used to correlate our results and obtain the converter sheet, the aim of the entire experiment.