Spike S2 Subunit: Possible Target for Detecting Novel SARS-CoV-2 Variants with Multiple Mutations

Area of Science:

• Virology
• Molecular Diagnostics
• Biotechnology

Background:

• Novel SARS-CoV-2 variants possess multiple mutations, potentially affecting the accuracy of molecular diagnostic assays.
• The Spike (S) protein's S2 subunit is highly conserved across SARS-CoV-2 variants, suggesting its utility as a stable diagnostic target.

Purpose of the Study:

• To evaluate the efficacy of the SARS-CoV-2 S2 subunit as a diagnostic target for novel variants.
• To assess the impact of incorporating S2 primers and probes into existing RT-PCR diagnostic kits.

Main Methods:

• Comparative analysis of 694 specimens using a commercial RT-PCR kit versus the same kit augmented with S2 primers and probes.
• Bioinformatic analysis of 230,821 SARS-CoV-2 nucleotide sequences to assess S2 primer and probe integrity against circulating variants.
• Evaluation of amplification efficiency and Ct values for S2 and other target genes (E, N, ORF1ab).

Main Results:

• No amplification issues were observed with the S2 target in any tested specimens, including the BA.4 Omicron variant.
• No statistically significant differences in mean Ct values were found between S2 and other target genes (E, N, ORF1ab) or between reagent batches.
• Bioinformatic analysis indicated a very low probability of performance issues due to mutations in S2 primers and probes across global SARS-CoV-2 variants.

Conclusions:

• The SARS-CoV-2 S2 subunit is a reliable and conserved target for molecular diagnostics, capable of detecting new variants.
• Integrating S2 detection into existing RT-PCR assays is feasible without compromising diagnostic sensitivity or specificity.
• The S2 subunit presents a promising strategy for developing robust diagnostic tools for current and future SARS-CoV-2 variants.