Next-Generation Sequencing (NGS) is a powerful technology that allows whole viral genome sequencing and is a crucial tool for identifying new mutations and variants. But NGS also has its limitations, including associated costs and relative low sequence capacity. Also, it can take 2-3 weeks before sequence data is available in public repositories. In the United States, only approximately 5% of SARS-CoV-2 positive samples sequences are analyzed, primarily by NGS.
Genotyping by polymerase chain reaction (PCR) can help scale up variant surveillance. Although genotyping by PCR does not analyze the complete viral genome as NGS does, it allows quicker results with shorter turnaround time of 12 to 24 hours. Genotyping by PCR is also less expensive and offers the possibility of higher testing throughput. It’s important to note that the broad availability of PCR instruments in laboratories worldwide supports global upscaling of variant surveillance.
Given the wide variety of existing and emerging variants, it is crucial to evaluate the overall performance of genotyping by PCR. The Variant Task Force (VTF), which is part of the National Institutes of Health’s (NIH’s) Rapid Acceleration of Diagnostics (RADxSM) initiative, initiated a project in July 2021 to identify SARS-CoV-2 markers that could detect all known variants. The study aimed to develop a panel of markers to accurately assign lineages and to support early detection of new and re-emerging variants by genotyping by PCR. The results of this study are published here.
SARS-CoV-2 detection by PCR using variant agnostic positivity markers
The study identified three variant agnostic makers to detect SARS-CoV-2 sequences, independent of lineage. The markers target sequences in the nsp10-, N- and S-genes. Under the criteria that a sample was considered positive for SARS-CoV-2 when at least one of the markers was detected, the study evaluated a total of 1,128 retrospective samples (1,031 SARS-CoV-2 positive and 97 SARS-CoV-2 negative samples). As a result, an outstanding performance with a Positive Percent Agreement (PPA) of 99.3% and a Negative Percent Agreement (NPA) of 100% was achieved.
“In this report, we demonstrated three (3) variant agnostic markers that can detect SARS-CoV-2 positive samples with high PPA and NPA compared to NGS.”
Genotyping by PCR supports monitoring of circulating variants
Using a total of 1,031 samples positive for SARS-CoV-2, the study assessed the performance of panels either consisting of 48, 24, 16, 12, or 8 makers to identify the top 10 WHO lineages (including Alpha, Beta, Gamma, Delta, Epsilon, Eta, Iota, Kappa, Lambda and Mu; but excluding the Omicron variant).
The results demonstrated that the 16 marker panel was sufficient to identify all 10 WHO lineages with a with a high PPA and NPA and results were similar to the 48-marker and 24-marker panels. The 12-marker panel and 8-marker panels identified eight and six of the WHO lineages respectively with high accuracy.
“……demonstrated that there are marker combinations that are highly specific for certain variants.”
Genotyping by PCR supports early detection of new variants
The study also explored the hypothesis that an increase in undetermined calls (when the marker panel is unable to associate a variant with a sample) could be indicative of new emerging variants. An increase in undetermined calls should therefore be regarded as a signal for focused full genome sequencing of those samples. Use of inexpensive, rapid PCR genotyping acts as filter to identify samples that should be NGS sequenced to identify a potential new variant.
A bioinformatics simulation with a modified 12-marker panel, which excluded the two Delta specific markers, was conducted using genomic SARS-CoV-2 data from the United States added to the Global Initiative on Sharing Avian Influenza Data (GISAID) database between November 2020 and July 2021. That period included an increase in Delta variant reports to GISAID from a few cases in April 2021 to >80% of all submitted sequences by July. The simulation revealed a direct correlation between prevalence data for the emerging Delta variant and increase in undetermined calls with the panel (without markers for Delta variant) starting in April and peaking in July 2021.
“Routine use of these genotyping markers could provide early warning that a new or re-emergent variant is circulating.”
Genotyping by PCR supports Omicron detection
To address the emerging Omicron variant, a panel of 4 markers was rapidly designed and tested. The panel included 3 Omicron and 1 Delta variant specific markers. Testing 1,631 SARS-CoV-2 positive samples with this new panel, this study resulted in 100% PPA and 99% NPA for the Omicron variant, and 90.9% PPA and 98.3% NPA for the Delta variant. It should be noted that the Delta samples that were not correctly identified were all Delta subtypes.
Following the discovery of the Omicron variant, the 4-marker panel was utilized in two CLIA-certified laboratories to genotype 5,372 SAR-CoV-2 positive samples during the month of December 2021. The results showed a surge in the relative prevalence of the Omicron variant in the United States, from about 15% to about 80% out of all genotyped positive samples from 9 December 2021 to 21 December 2021. Subsequently, this panel was modified to include a marker to differentiate BA.1 from BA.2/BA.3 sub-lineages of Omicron and to date more that 75,000 samples have been genotyped with representation of samples from all 50 states within the US.
In summary, according to the authors, genotyping by PCR is quick, efficient, and less expensive than sequencing in SARS-CoV-2 surveillance, and therefore it provides an excellent tool for scaling up surveillance efforts in this ongoing pandemic. The authors emphasize that any sample that cannot be determined by genotyping by PCR would be a prime candidate for sequencing. An increase in undetermined samples could signal the presence of a new emerging variant, and genotyping by PCR offers the potential of an early warning system.
“This illustrates the symbiotic nature of using genotyping markers in conjunction with targeted sequencing.”
This work was supported by a collaboration between the NIH Rapid Acceleration of Diagnostics (RADx), Helix, University of Washington, Aegis Biosciences, Rosalind and Thermo Fisher Scientific. SARS-CoV-2 variant information generated from this program is available to the public through the Rosaland website. The Rosalind Tracker dashboard provides a snapshot of variants circulating in the United States close to real-time.
For more information on the assays associated with the publication, please visit:
Assays for the following mutations used in this study are available from Thermo Fisher:
A method for variant agnostic detection of SARS-CoV-2, rapid monitoring of circulating variants, detection of mutations of biological significance, and early detection of emergent variants such as Omicron by E. Lai, D. Becker, et al
* For research use only. Not for use in diagnostic procedures.