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

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.

SARS-CoV-2 genomic surveillance and reliability of PCR single point mutation assay (SNPsig® SARS-CoV-2 EscapePLEX CE) for the rapid detection of variants of concern in Cameroon

“…As an alternative to sequencing, the SNPsig® SARS-CoV-2 EscapePLEX CE Kit has received much attention as a genotyping test for the rapid detection and identification of SARS-CoV-2 VOCs [ 32 , 33 ]. Our analyses showed an overall good concordance with Sanger sequencing in discriminating between variants and wild-type strains ( Table 3 ), thus suggesting its suitability for preliminary identification of VOCs in Cameroon.…”

RT-PCR/MALDI-TOF Diagnostic Target Performance Reflects Circulating SARS-CoV-2 Variant Diversity in New York City

“…1 Therefore, other than a finite number of mutations in the spike protein (eg, N501Y, S477N, V1176F, N501Y), one can anticipate that other variant-defining polymorphisms may not persist over continued circulation of SARS-CoV-2 and, thus, new target formulations will need to be continuously considered. As most efforts to accommodate new variants have been to redesign primers and probes or include more targets in this region, 37,44 this may become impractical as inclusion of additional reactions can become costly and introduce more room for error. Furthermore, although multiplexing assays allow for single-pot reactions, the level of target redundancy is limited by the optical detection system as more fluorophores complicate conventional diagnostic methods.…”

Wastewater monitoring using a novel, cost-effective PCR-based method that rapidly captures the transition patterns of SARS-CoV-2 variant prevalence (from Delta to Omicron) in the absence of conventional surveillance evidence

“…As Delta is the currently predominant variant worldwide, the assay's design was focused to enable preferable amplification of Omicron VOC targets and minimize to the highest degree possible the amplification of Delta (along with Beta and Gamma that present similar characteristics at the respective upstream primer-hybridization regions). Additional primer sequence analysis was performed using the DINAMelt software [25] to estimate, at the annealing temperature of 58 °C, the mole fraction the OmUp primer hybridized to the target sequences of the designated VOCs [12,13,24]. Α 6-carboxyfluorescein (FAM)-conjugated locked nucleic acid (LNA) TaqMan probe (OmProbe: 5'-FAM-ACTT+CTC+A+A+TGGAA+GC-IBFQ-3', Tm: 62.5 °C) was designed to enable fluorescence detection.…”

“…The length of the probe was also kept short (16 nucleotides) to improve mismatch discrimination. The philosophy behind the design of LNA TaqMan probes for SARS-CoV-2 VOC typing based on RT-PCR-based SNP identification has been presented in detail in our previous works [12,13]. Via this approach, discrimination between the Omicron and the Alpha is feasible on the bases of the obtained fluorescence.…”

“…Consequently, alternative methodologies for the rapid detection of mutations and VOCs have been proposed by the ECDC, including the use of RT-PCR-based assays [10]. So far, such real-time RT-PCR-based methodologies were developed by our team to facilitate identification of critical SNPs associated with Alpha, Beta/Gamma and Delta VOCs, and the performance of the assays was subsequently evaluated in previously characterized human and veterinary clinical specimens [12,13].…”