A comparative analysis exposes an amplification delay distinctive to SARS-CoV-2 Omicron variants of clinical and public health relevance

ABSTRACT

Mutations in the SARS-CoV-2 genome may negatively impact a diagnostic test, have no effect, or turn into an opportunity for rapid molecular screening of variants. Using an in-house Emergency Use Authorized RT-qPCR-based COVID-19 diagnostic assay, we combined sequence surveillance of viral variants and computed PCR efficiencies for mismatched templates. We found no significant mismatches for the N, E, and S set of assay primers until the Omicron variant emerged in late November 2021. We found a single mismatch between the Omicron sequence and one of our assay’s primers caused a > 4 cycle delay during amplification without impacting overall assay performance.

Starting in December 2021, clinical specimens received for COVID-19 diagnostic testing that generated a Cq delay greater than 4 cycles were sequenced and confirmed as Omicron. Clinical samples without a Cq delay were largely confirmed as the Delta variant. The primer-template mismatch was then used as a rapid surrogate marker for Omicron. Primers that correctly identified Omicron were designed and tested, which prepared us for the emergence of future variants with novel mismatches to our diagnostic assay's primers. Our experience demonstrates the importance of monitoring sequences, the need for predicting the impact of mismatches, their value as a surrogate marker, and the relevance of adapting one's molecular diagnostic test for evolving pathogens.

KEYWORDS:

• Omicron variant
• SARS-CoV-2
• Delta variant
• RT-qPCR
• diagnosis
• mismatch
• amplification efficiency
• COVID-19

Acknowledgements

The authors thank all members of the Molecular Diagnostics Laboratory at the Fralin Biomedical Research Institute for feedback and technical support. The authors would also like to thank Dr. J. Webster for comments and manuscript editing. This project was supported by the Department of General Services of the Commonwealth of Virginia (DGS-201020-UVT) and funds from the Fralin Biomedical Research Institute at VTC.

We gratefully acknowledge all data contributors, including authors and their originating laboratories responsible for obtaining the specimens, and the submitting laboratories for generating the genetic sequence and metadata and sharing via the GISAID Initiative, on which this research is based. K.B. and C.V.F. conceived the idea of the manuscript. K.B. wrote the first draft, performed the viral genomic surveillance studies summarized in Figure 1, and analyzed the data presented in Tables 1 and 2, Supplementary Data 1, and Figure 2 and 5. A.C. analyzed the data summarized in Figures 2, 3A, 3D, 4, 5, 6 and Supplementary Figure 2. C.R. and R.B. carried out the experiments summarized in Figures 2 and 5 and analyzed all RMA sequencing data summarized in Figure 3. D.Z. acted as our CLIA consultant. R.K., R.M., S.T.K., and D.T. carried out the Center study summarized in Figure 3D. M.F. contributed to the implementation of the research. All authors aided in interpreting results. C.V.F. designed all figures, analyzed WGS sequences, and wrote the final paper with input from all authors. C.V.F. supervised the project.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by Fralin Biomedical Research Institute [Grant Number N/A]; Department of General Services of the Commonwealth of Virginia [Grant Number DGS-201020-UVT].