Ensuring our diagnostic assays detect the Omicron variant

Updated : Fri, December 10, 2021 @ 2:34 AM

On 22 November 2021, The Network for Genomics Surveillance in South Africa detected a new SARS-CoV-2 variant, named the B.1.1.529 lineage.¹ By 26 November 2021, the World Health Organization (WHO) had already designated it a variant of concern, named Omicron, due to its large number of mutations, some with worrying characteristics.² Omicron has a total of over 50 mutations, with more than 30 of these found within the spike protein³ leading to concerns regarding immune escape and the potential for increased transmissibility.⁴ A preprint of a study of SARS-CoV-2 reinfection in South Africa suggests that in contrast to Beta and Delta variants, there is substantial evidence that Omicron can evade immunity from prior infection.⁵ Scientists around the world are working to characterise the transmissibility of the variant, the severity of the disease it causes and to determine the effect it will have on counter-measures, such as vaccines, diagnostics and treatments. Omicron has now been reported in many countries worldwide, including by community transmission. Good global surveillance is needed to track the spread of this new variant, as well as to continue to monitor the Delta variant, which remains the predominant variant circulating globally.⁶ This requires a combination of PCR testing, genotyping and sequencing.

A closer look at the mutations

The SARS-CoV-2 virus uses its spike protein to bind to host cellular receptors. The receptor binding domain (RBD) is a key functional component of the spike protein that binds to angiotensin-converting enzyme 2 (ACE2) receptors, leading to cell entry.⁷

The Omicron variant has over 30 mutations in its spike protein, including amino acid substitutions, deletions and an insertion. They are as follows (with substitutions in the RBD in bold type):

A67V, 69-70del, T95I, 143-145del, 211del, 214insEPE, G339D, S371L, S373P, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F.^3,8

Some of these mutations identified in the Omicron variant are also present in other variants of concern. Analysis of the mutations in the spike protein indicates they may be associated with changes in transmissibility or immune escape.⁸

There are also a number of additional mutations throughout the genome, including two substitutions and a deletion in the N gene and one substitution in the E gene, all resulting in changes to the amino acid sequence.³ Both the N and E genes are widely used as targets for diagnostic assays.

Impact analysis of Omicron mutations on the CDC N1 assay

With any mutation comes the risk that diagnostic assays may no longer perform as expected. As detailed in our October 2021 technical bulletin, LGC, Biosearch Technologies™ has a four-tier assay screening and replacement programme to identify and mitigate against the risk of assay impairment in the event of a mutation arising within the target binding site of diagnostic assays.

1. Assays are monitored at fortnightly intervals. This includes the N1 and N2 assays which are components of our ultra-high-throughput SARS-CoV-2 testing systems, 2019-nCoV ValuPanel™ Reagents, and the SC2 assay (N gene) which is a component of the Influenza SARS-CoV-2 Multiplex ValuPanel Reagents.
2. Each of these sequences is compared to published sequences that are reported in the GISAID database.
3. Any mutations of concern are subjected to further in silico analysis and wet lab verification if required.
4. Additional assays have been evaluated as ready for deployment, should a detrimental mutation be identified. These additional assays (including the E sarbeco assay) are also under constant surveillance.

As of 29 November 2021 we have evaluated the 144 B.1.1.529 sequences on GISAID database. We can confirm based on these sequences the following with respect to the N1, N2 and E sarbeco assays:

• a single base change (C-T transition) in position 3 (5-3 prime) of the N1 probe.  
• a single base change (C-T transition) in position 2 (5-3 prime) of the E Sarbeco forward primer.
• no impact on any other oligonucleotide targeting N1, N2 or E sarbeco.

In silico analysis reveals these Omicron mutations (P13L in the N gene and T9I in the E gene) are unlikely to impact the performance of the assays but we are in the process of conducting laboratory evaluations to confirm this.

The N gene mutation (P13L) is also present in the Lambda variant. Discussions with other diagnostic laboratories have indicated that this mutation does not affect the performance of CDC N1 diagnostic assay.

Using SARS-CoV-2 Variant ValuPanel assays for genotyping Omicron

SARS-CoV-2 Variant ValuPanel assays* are designed for qualitative detection of specific SARS-CoV-2 mutations by genotyping using reverse transcription-polymerase chain reaction (RT-PCR). Each SARS-CoV-2 Variant ValuPanel consists of 2 BHQplus™ Probes and 2 primers for amplification and simultaneous discrimination between a specific mutation and the wild-type SARS-CoV-2 sequence. The assays are designed for variant screening in respiratory tract samples that have previously tested positive for SARS-CoV-2 by diagnostic RT-PCR. 

To differentiate Omicron (B.1.1.529) from other variants of concern, we recommend using an array of SARS-CoV-2 Variant ValuPanel assays, including those for 69-70 Del (present in the Omicron BA.1 lineage) and K417N (present in both Omicron BA.1 and BA.2 lineages).

What we’re doing next

Wet lab testing is underway to assess the performance of our assays against Omicron genetic material. In accordance with our standard practice, we continue to perform in silico analysis of our probe and primer sequences against newly published sequences that are reported in the GISAID database. We will remain vigilant to ensure our assays remain functional and will not hesitate to deploy a new assay should we discover that a compromising mutation has arisen. Stay up to date on how we're monitoring variants here.

*For Research Use Only. Not for use in diagnostic procedures.

References

1. SA National Institute for Communicable Diseases. Frequently asked questions for the B.1.1.529 mutated SARS-CoV-2 lineage in South Africa. https://www.nicd.ac.za/frequently-asked-questions-for-the-b-1-1-529-mutated-sars-cov-2-lineage-in-south-africa/. Published November 26, 2021. Accessed December 6, 2021.
2. World Health Organization. Classification of Omicron (B.1.1.529): SARS-CoV-2 Variant of Concern. https://www.who.int/news/item/26-11-2021-classification-of-omicron-(b.1.1.529)-sars-cov-2-variant-of-concern. Published November 26, 2021. Accessed December 6, 2021.
3. UK Health Security Agency. SARS-CoV-2 variants of concern and variants under investigation in England. Variant of concern: Omicron, VOC-21NOV-01 (B.1.1.529). Technical briefing 30. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/1038404/Technical_Briefing_30.pdf. Published December 3, 2021. Accessed December 7, 2021.
4. European Centre for Disease Prevention and Control. Threat Assessment Brief: Implications of the emergence and spread of the SARS-CoV-2 B.1.1. 529 variant of concern (Omicron) for the EU/EEA. https://www.ecdc.europa.eu/en/publications-data/threat-assessment-brief-emergence-sars-cov-2-variant-b.1.1.529. Published November 26, 2021. Accessed December 7, 2021.
5. Pulliam JRC, van Schalkwyk C, Govender N, von Gottberg A, Cohen C, Groome MJ, Dushoff J, Mlisana K, Moultrie H. Increased risk of SARS-CoV-2 reinfection associated with emergence of the Omicron variant in South Africa. medRxiv. https://www.medrxiv.org/content/10.1101/2021.11.11.21266068v2.full.pdf. Published December 2, 2021. Accessed December 7,2021.
6. Hodcroft EB. CoVariants: SARS-CoV-2 Mutations and Variants of Interest. https://covariants.org/. Published 2021. Accessed December 7, 2021.
7. Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, Zhang Q, Shi X, Wang Q, Zhang L, Wang X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 581, 215–220. https://doi.org/10.1038/s41586-020-2180-5. Published 2020. Accessed December 7, 2021.
8. US Centers for Disease Control and Prevention. Science Brief: Omicron (B.1.1.529) Variant. https://www.cdc.gov/coronavirus/2019-ncov/science/science-briefs/scientific-brief-omicron-variant.html. Published December 2, 2021. Accessed December 7, 2021.