A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain

Serafeim C Chaintoutis,Taxiarchis Chassalevris,Lemonia Skoura,George Tsiolas,Anagnostis Argiriou,Evangelia Mouchtaropoulou,Chrysostomos I Dovas,Areti Tychala,Ioannis Vlatakis,Sofia Balaska,Dimitris Koutsioulis

doi:10.3390/life11101015

Abstract

Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD. This region contains most contacting residues of SARS-CoV-2 that bind to ACE2. A novel strategy employing the use of non-extendable LNA oligonucleotide blockers that can reduce non-specific hybridization of probes increased the number of different mutated sites examined in a multiplex PCR. The combinatory analysis of the different fluorescence signals obtained enabled the preliminary differentiation of SARS-CoV-2 variants of concern. The assay is sensitive with a LOD of 263 copies/reaction for the Delta variant, 170 copies/reaction for the Beta variant, amplification efficiencies > 91% and a linear range of >5 log10 copies/reaction against all targets. Validation of the assay using known SARS-CoV-2-positive and negative samples from humans and animals revealed its ability to correctly identify the targeted mutations and preliminary characterize the SARS-CoV-2 variants. The novel approach for mutation typing using LNA oligonucleotide blockers can be modified to target signature mutations at four different sites in the RBM and further expand the range of variants detected.

Highlights

The continuous emergence of mutations resulting to amino-acid substitutions in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of major importance
Aim of this study was to develop a novel real-time RT-PCR-based methodology able to identify signature mutations present in more than two sites of the viral receptorbinding motif (RBM) that contains most of the contacting residues of SARS-CoV-2 that bind to ACE2
The analytical parameters of the developed assay were determined as follows: RNA extracts from two SARS-CoV-2-positive nasopharyngeal clinical samples, i.e., one Delta variant strain and one Beta variant strain were quantified using CDC’s N2 real-time RT-PCR, as described previously [18]

Summary

Introduction

The continuous emergence of mutations resulting to amino-acid substitutions in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is of major importance. A novel S:T478K (threonine to lysine) substitution in the RBD is so far a unique characteristic of Delta variant strains [6], that since January 2021 has been found in increasing frequencies in several countries [7] This variant is characterized by decreased sensitivity to antibody-mediated neutralization [8], immediate identification for its presence in infected individuals is critical, as is for the three aforementioned VOCs. Beyond the described VOCs, other SARS-CoV-2 variants, which have been designated as variants under monitoring or variants under investigation are of importance. Aim of this study was to develop a novel real-time RT-PCR-based methodology able to identify signature mutations present in more than two sites of the viral receptorbinding motif (RBM) that contains most of the contacting residues of SARS-CoV-2 that bind to ACE2 This was feasible by employing non-extendable locked nucleic acid (LNA) oligonucleotides that reduce non-specific hybridization of probes. Based on the combinatory interpretation of the obtained fluorescence signals, the developed assay enables the preliminary characterization of VOCs and other SARS-CoV-2 variants under investigation or monitoring with potential public health impact

Materials and Methods

Real-Time RT-PCR

Analytical Characteristics

Specificity and Diagnostic Performance

Results