The rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories.
Background
Mutations in the receptor binding domain of the SARS-CoV-2 Spike protein are associated with increased transmission or substantial reductions in vaccine efficacy, including in the recently described Omicron variant. The changing frequencies of these mutations combined with their differing susceptibility to available therapies have posed
significant problems for clinicians and public health professionals.
Objective
To develop an assay capable of rapidly and accurately identifying variants including Omicron in clinical specimens to enable case tracking and/or selection of appropriate clinical treatment.
Study Design
Using three duplex RT-ddPCR reactions targeting four amino acids, we tested 419 positive clinical specimens from February to December 2021 during a period of rapidly shifting variant prevalences and compared genotyping results to genome sequences for each sample, determining the sensitivity and specificity of the assay for each variant.
Results
Mutation determinations for 99.7% of detected samples agree with NGS data for those samples, and are accurate despite wide variation in RNA concentration and potential confounding factors like transport medium, presence of additional respiratory viruses, and additional mutations in primer and probe sequences. The assay accurately
identified the first 15 Omicron variants in our laboratory including the first Omicron in Washington State and discriminated against S-gene dropout Delta specimen.
Conclusion
We describe an accurate, precise, and specific RT-ddPCR assay for variant detection that remains robust despite being designed prior the emergence of Delta and Omicron variants. The assay can quickly identify mutations in current and past SARS-CoV-2 variants, and can be adapted to future mutations.
The rapid emergence of new SARS-CoV-2 variants raises a number of public health questions including the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to better understand patterns of transmission and possible load on healthcare resources. Next-Generation Sequencing (NGS) is the primary method for detecting and tracing the emergence of new variants, but it is expensive, and it can take weeks before sequence data is available in public repositories. Here, we describe a Polymerase Chain Reaction (PCR)-based genotyping approach that is significantly less expensive, accelerates reporting on SARS-CoV-2 variants, and can be implemented in any testing lab performing PCR. Specific Single Nucleotide Polymorphisms (SNPs) and indels are identified that have high positive percent agreement (PPA) and negative percent agreement (NPA) compared to NGS for the major genotypes that circulated in 2021. Using a 48-marker panel, testing on 1,128 retrospective samples yielded a PPA and NPA in the 96.3 to 100% and 99.2 to 100% range, respectively, for the top 10 most prevalent lineages. The effect on PPA and NPA of reducing the number of panel markers was also explored. In addition, with the emergence of Omicron, we also developed an Omicron genotyping panel that distinguishes the Delta and Omicron variants using four (4) highly specific SNPs. Data from testing demonstrates the capability to use the panel to rapidly track the growing prevalence of the Omicron variant in the United States in December 2021.
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