Several SARS-CoV-2 variants of concern (VOC) have emerged. To be able to confirm infection with a specific variant, sequencing of the whole SARS-CoV-2 genome, or at least whole or partial S-gene for the current variants is required.

While countries are building or up-scaling their high throughput sequencing capacities, results are made available with a time delay that makes Whole genome sequencing (WGS) insufficient for timely detection of variants for public health response (e.g. contact tracing) and calculation of prevalence of VOCs in the community. Despite a significant drop of costs, WGS is still a relatively expensive method in comparison with some of the screening approaches based on PCR. Sanger sequencing of the S-gene, can in some settings be more feasible and timely than WGS.

For early detection and prevalence calculation of VOCs (i.e. B.1.1.7/501Y.V1, B.1.351/501Y.V2, P.1/501Y.V3), alternative methods, such as using diagnostic screening PCR-based assays that generate results in a few hours, with subsequent verification/confirmation by sequencing, can be valuable.

Sequencing

Whole genome sequencing

Whole genome sequencing is an important method to characterise viruses genetically. Using either a tiled amplicon approach or shotgun sequencing, the entire genome of the virus will be sequenced and can be compared with other circulating strains [1]. Whole genome sequencing can be used efficiently to detect VOCs as it represents an unbiased approach without the need for prior knowledge on the presence of certain mutations in the viral genome. It is a resource-intensive method that can take several days for generation of results, depending on the protocol.

Sanger or partial next generation sequencing ampliconbased sequencing

Sanger or next generation sequencing (NGS) amplicon-based sequencing of selected parts of the viral genome are alternative methods for the identification of VOCs. With these techniques, targeted whole or partial S-gene sequencing can be performed using a genetic analyser. The NGS method comes with the same challenges as WGS regrading equipment and bioinformatics analysis. Protocols for specific RT-PCRs for marker regions of the S-gene region indicative of the B.1.1.7/501Y.V1 and B1.351/501Y.V2 VOCs, followed by sequencing have been developed [3]. The region to be sequenced should cover at least the entire N-terminal and receptor binding domain (RBD) (amino acid 1-541, 1623 bp) to reliably differentiate between the circulating variants.

Diagnostic screening assays of known VOCs

S-gene drop out or target failure

For the B.1.1.7/501Y.V1 (also called VOC 202012/01), a negative or significantly weaker positive S-gene result in multiplex RT-PCR assays, with positive results for the other targets, has been used as an indicator or screening method to identify this particular variant. The weaker signal or complete failure of the S-gene target is caused by a deletion at nt207-212 in the respective gene.

Multiplex RT-PCR, including S-gene target failure

With a multiple channel real time RT-PCR device, the normal E and/or N and/or ORF-1 target assays may be combined with the S-gene target, so the VOC screening could be integrated with the normal routine, in a single run [7].

It is important to emphasise that results should not be over-interpreted and must be checked/continuously validated through the use of genomics.

Screening SNP assays

Screening for VOC specific amino acid substitutions can be done using a specific RT-PCR assays targeting single nucleotide polymorphisms (SNP) to screen e.g. spike N501Y and HV69-70del mutations (e.g. present in B.1.1.7/501Y.V1 VOC) [7]. Appropriate positive controls will be needed.

Screening SNP by specific real time RT-PCR melting curve analysis

Some real time PCR platforms allow for melting curve analysis. Commercial assays have been developed to use this genotyping method to identify specific amino acid substitutions.

Reverse transcription loop-mediated and transcriptionmediated amplification isothermal amplification

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) and transcription-mediated amplification (TMA) on Panther Hologic machines techniques have emerged as an alternative molecular detection method for the detection of SARS-CoV-2. RT-LAMP technique has some advantages such as faster test results and need of fewer resources, while maintaining high sensitivity and specificity, although currently available protocols will not differentiate between specific VOCs [9].

Rapid antigen detection tests

Rapid antigen tests can contribute to overall COVID-19 testing capacity, offering advantages in terms of shorter turnaround times and reduced costs, especially in situations in which RT-PCR testing capacity is limited, although their sensitivity is generally lower than for RT-PCR [10]. Rapid antigen tests may detect the presence of SARSCoV-2 (including variant viruses) but cannot identify/differentiate the type of VOC.

The need to remain vigilant

As SARS-CoV-2 is an RNA virus and mutates with intermediate frequency, it is expected that more VOCs will emerge. Genomics is the best tool for identification of new variants. The diagnostic laboratories need to remain vigilant to detect any mismatches of RT-PCR assay primers and probes in comparison to circulating virus genomes and detection capability of other assays such as rapid antigen tests, and to adapt Sanger sequencing protocols. The vast majority of primer/probe binding sites of commercial assays are not publicly known. It is important to note that it was coincidental that detection assays targeting the S-gene enable the identification of B.1.1.7 lineage variants. For all assays, it is vital to keep track of possible incidents of suboptimal performance and to inform the manufacturer of a commercial assay and international SARS-CoV-2 public health networks of any concerns you may experience with a specific assay.

Abstract from: Methods-for-the-detection-identification-of-SARS-CoV-2-variants-WHO-ECDC

References
1. European Centre for Disease Prevention and Control. Sequencing of SARS-CoV-2: first update 2021.  Available from: https://www.ecdc.europa.eu/sites/default/files/documents/Sequencing-of-SARS-CoV-2-first-update.pdf 
3. Université de Genève and Hôpitaux Universitaires de Genève. Protocol for specific RT-PCRs for marker regions of the Spike region indicative of the UK SARS-CoV-2 variant B.1.1.7 and the South African variant 501Y.V2 2021. Available from: https://www.hug.ch/sites/interhug/files/structures/laboratoire_de_virologie/protocol_amplification_voc_20201 201_uk_geneva.pdf 
7. Gulay Korukluoglu, Kolukirik M, Bayrakdar F, Ozgumus GG, Altas AB, Cosgun Y, et al. 40 minutes RT-qPCR Assay for Screening Spike N501Y and HV69-70del Mutations 2021. Available from: https://www.biorxiv.org/content/10.1101/2021.01.26.428302v1.full.pdf
9. Dao Thi VL, Herbst K, Boerner K, Meurer M, Kremer LP, Kirrmaier D, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Science Translational Medicine. 2020;12(556):eabc7075.
10. European Centre for Disease Prevention and Control. Options for the use of rapid antigen tests for COVID-19 in the EU/EEA and the UK 2020. Available from: https://www.ecdc.europa.eu/en/publicationsdata/options-use-rapid-antigen-tests-covid-19-eueea-and-uk