Detection of SARS-CoV-2 Variants via Different Diagnostics Assays Based on Single-Nucleotide Polymorphism Analysis

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is characterized by fast evolution with the appearance of several variants. Next-Generation Sequencing (NGS) technology is considered the gold standard for monitoring known and new SARS-CoV-2 variants. However, the complexity of this technology renders this approach impracticable in laboratories located in areas with limited resources. We analyzed the capability of the ThermoFisher TaqPath COVID-19 RT-PCR (TaqPath) and the Seegene Novaplex SARS-CoV-2 Variant assay (Novaplex) to detect Omicron variants; the Allplex VariantII (Allplex) was also evaluated for Delta variants. Sanger sequencing (SaS) was the reference method. The results obtained with n = 355 nasopharyngeal samples were: negative with TaqPath, although positive with other qualitative molecular assays (n = 35); undetermined (n = 40) with both the assays; negative for the ∆69/70 mutation and confirmed as the Delta variant via SaS (n = 100); positive for ∆69/70 and confirmed as Omicron BA.1 via SaS (n = 80); negative for ∆69/70 and typed as Omicron BA.2 via SaS (n = 80). Novaplex typed 27.5% of samples as undetermined with TaqPath, 11.4% of samples as negative with TaqPath, and confirmed 100% of samples were Omicron subtypes. In total, 99/100 samples were confirmed as the Delta variant with Allplex with a positive per cent agreement (PPA) of 98% compared to SaS. As undermined samples with Novaplex showed RdRp median Ct values (Ct = 35.4) statistically higher than those of typed samples (median Ct value = 22.0; p < 0.0001, Mann-Whitney test), the inability to establish SARS-CoV-2 variants was probably linked to the low viral load. No amplification was obtained with SaS among all 35 negative TaqPath samples. Overall, 20% of samples which were typed as negative or undetermined with TaqPath, and among them, twelve were not typed even by SaS, but they were instead correctly identified with Novaplex. Although full-genome sequencing remains the elected method to characterize new strains, our data show the high ability of a SNP-based assay to identify VOCs, also resolving samples typed as undetermined with TaqPath.

Keywords: SARS-CoV-2; SARS-CoV-2 variant assay; SARS-CoV-2 variants; molecular diagnosis; single-nucleotide polymorphism (SNP).

Figure 1

SARS-CoV2 S gene and SNP detected via Novaplex and Allplex assays. SNPs detected are reported in blue color, protein domains where SNPs lay are depicted in green. NTD, N-terminal domain; RDB, receptor-binding domain; TM, transmembrane domain.

Figure 2

SARS-CoV-2 variants found during the observation period. Variant stacked plot indicating the distribution of SARS-CoV-2 variants among samples collected at INMI from December 2021 to March 2022. Blue and orange areas show results obtained using TaqPath COVID-19 RT-PCR kit; lines show identification of SARS-CoV-2 variants via Sanger sequencing.

Figure 3

Flow-chart of SARS-CoV-2 variant analysis carried out in SARS-CoV-2-positive NPSs tested with TaqPath. Both SaS and Novaplex assays were used to analyze undetermined and negative TaqPath samples. AllplexVariant II was used to confirm Delta variant. Neg, negative; NO AMP, no amplification; Und, undetermined.

Figure 4

Comparison of Ct values of E484A, N501Y, and RdRp for samples that were undetermined or negative with TaqPath and typed using Novaplex.

Figure 5

Samples untyped using TaqPath and Novaplex assays. Comparison of number of samples classed as undetermined and negative with both assays.