Journal Article

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

Rueca, Martina, Fabeni, Lavinia, Minosse, Claudia, Giombini, Emanuela, Amendola, Alessandra, Garbuglia, Anna Rosa, Specchiarello, Eliana, Gruber, Cesare Ernesto Maria, Carletti, Fabrizio, Matusali, Giulia, Maggi, Fabrizio

2023

Overview

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.

• Publisher: MDPI AG,MDPI
• Subject: Coronaviruses / COVID-19 / DNA sequencing / Gene amplification / Genetic aspects / Genomics / Health aspects / Laboratories / molecular diagnosis / Mutation / Nucleotide sequencing / Public health / RNA polymerase / SARS-CoV-2 / SARS-CoV-2 variant assay / SARS-CoV-2 variants / Severe acute respiratory syndrome / Severe acute respiratory syndrome coronavirus 2 / Single nucleotide polymorphisms / single-nucleotide polymorphism (SNP) / Software