SARS-CoV-2 Subgenomic N (sgN) Transcripts in Oro-Nasopharyngeal Swabs Correlate with the Highest Viral Load, as Evaluated by Five Different Molecular Methods

Abstract

The COVID-19 pandemic has forced diagnostic laboratories to focus on the early diagnostics of SARS-CoV-2. The positivity of a molecular test cannot respond to the question regarding the viral capability to replicate, spread, and give different clinical effects. Despite the fact that some targets are covered by commercially-available assays, the identification of new biomarkers is desired in order to improve the quality of the information given by these assays. Therefore, since the subgenomic transcripts (sgN and sgE) are considered markers of viral activity, we evaluated these subgenomic transcripts in relation to the genomic amplification obtained using five different commercial CE-IVD tools. Methods: Five CE-IVD kits were compared in terms of their capability to detect both synthetic SARS-CoV-2 viral constructs (spiked in TMB or PBS medium) and targets (N, E, RdRp and Orf1ab genes) in twenty COVID-19-positive patients' swabs. The sgN and sgE were assayed by real-time RT-qPCR and digital PCR. Results: None of the diagnostic kits missed the viral target genes when they were applied to targets spiked in TMB or PBS (at dilutions ranging from 100 pg to 0.1 pg). Nevertheless, once they were applied to RNA extracted from the patients' swabs, the superimposability ranged from 50% to 100%, regardless of the extraction procedure. The sgN RNA transcript was detected only in samples with a higher viral load (Ct ≤ 22.5), while sgE was within all of the Ct ranges. Conclusions: The five kits show variable performances depending on the assay layout. It is worthy of note that the detection of the sgN transcript is associated with a higher viral load, thus representing a new marker of early and more severe infection.

Keywords: CE-IVD; SARS-CoV-2 subgenomic regions; viral load.

Figure 1

Spike-in of genes N (a) and E (b) evaluated by Kit-A. Figures (a,b) show the regression plots regarding the correlation curves obtained by the real-time TaqMan PCR Kit-A on serial dilutions of both synthetic positive control (pcs; copies of target) genea N and E. The starting volume of the input RNA for the reverse transcription and qPCR (one step process) was 5 µL for each target. The RNA amounts corresponding to each titration point amplified by one-step RT-qPCR were 0.1 pg, 1 pg,10 pg, 100 pg, and 1 ng for both the N and E genes, respectively. The reference sequences for the primers are reported in Table 3.

Figure 2

Melting curve profiles obtained by HRMA on the different genomic and subgenomic regions of SARS-CoV-2. The amplification plots of the three targets are clearly shown, as the melting temperatures (Tm) are different: Gene N (Tm = 86.5), sgN (Tm = 82.5), and sgE (78.0), respectively.

Figure 3

Analysis by ddPCR analysis on samples with low Ct (≤22.5) for N, sgE and sgN transcripts. ddPCR analysis output regarding samples with either Ct ≤ 22.5 or Ct > 22.5 in the realtime PCR analysis. Gene N is present in all of the samples with Ct below or above the 22.5 value. The sgN RNA is only detectable for Ct ≤ 22.5, while the sgE transcript is within all of the of the Ct ranges.