key: cord-1007414-hrxdj4rw
authors: Pabbaraju, Kanti; Zelyas, Nathan; Wong, Anita; Croxen, Matthew A.; Lynch, Tarah; Buss, Emily; Murphy, Stephanie; Shokoples, Sandy; Kanji, Jamil; Tipples, Graham
title: Evolving strategy for an evolving virus: development of real-time PCR assays for detecting all SARS-CoV-2 variants of concern
date: 2022-05-26
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2022.114553
sha: 6cca66aa8377b8ce6345014ff0561a30fd5052ea
doc_id: 1007414
cord_uid: hrxdj4rw

In order to detect the SARS-CoV-2 variants of concern (VOCs), five real-time reverse transcriptase PCR (rRT-PCR) assays were designed to target the critical discriminatory mutations responsible for the following amino acid changes in the spike protein: two Δ69-70 + N501Y + E gene triplexes (one optimized for Alpha [B.1.1.7] and one optimized for Omicron [B.1.1.529]), a K417N + 242-244 wild-type duplex, a K417T + E484K duplex, and a L452R + P681 + E484Q triplex. Depending on the assay, sensitivity was 98.97-100% for the detection of known VOC-positive samples, specificity was 97.2-100%, limit of detection was 2-116 copies/reaction, intra- and interassay variability was less than 5%, and no cross-reactivity with common respiratory pathogens was observed with any assay. A subset of rRT-PCR- positive VOC samples were further characterized by genome sequencing. A comparison of the lineage designation by the VOC rRT-PCR assays and genome sequencing for the detection of the Alpha, Beta, Gamma, Delta and Omicron variants showed clinical sensitivities of 99.97% to 100%, clinical specificities of 99.6% to 100%, positive predictive values of 99.8% to 100%, and negative predictive values of 99.98% to 100%. We have implemented these rRT-PCR assays targeting discriminatory single nucleotide polymorphisms for ongoing VOC screening of SARS-CoV-2 positive samples for surveillance purposes. This has proven extremely useful in providing close to real-time molecular surveillance to monitor the emergence of Alpha, the replacement of Alpha by Delta, and the replacement of Delta by Omicron. While the design, validation and implementation of the variant specific PCR targets is an ever-evolving approach, we find the turn-around-time, high throughput and sensitivity to be a useful complementary approach for SARS-CoV-2 genome sequencing for surveillance purposes in the province of Alberta, Canada.

The World Health Organization (WHO) case definition for SARS-CoV-2 variants of concern (VOCs) includes lineages with increased transmissibility or detrimental change in COVID-19 epidemiology, increased virulence or change in clinical disease presentation, or decreased effectiveness of public health and social measures or available diagnostics, vaccines, and therapeutics (1) (7) countries; the spread of Delta was faster than prior VOCs and it was reported in 54 countries by 1 June 2021 (8) . The rate of spread for Omicron surpassed all previous VOCs and as of 6 January 2022, Omicron had been identified in 149 countries across all six WHO Regions (9) .

Alpha has 13 mutations in the spike (S) protein with the Δ69-70, N501Y and P681H mutations contributing to its increased transmissibility (10) . Gamma displays additional critical mutations including K417T, E484K, N501Y, L18F, T20N, P26S, D138Y, R190S, and H655Y (amino acids located in the receptor-binding, N-terminal or furin cleavage domains of the spike protein), and has been shown to be more resistant to multiple neutralizing monoclonal antibodies and vaccine antisera J o u r n a l P r e -p r o o f (11) . Beta also displays mutations at the same three receptor-binding domain residues as Gamma with K417N, E484K, and N501Y, which could reduce monoclonal antibody or vaccine efficacy (3, 12) . These residues are also associated with increased affinity to the human ACE2 receptor that can impact host cell entry and virus transmission (13) . Important changes in the spike protein for Delta include T19R, Δ157-158, L452R, T478K, D614G, P681R, and D950N (14) ; L452R and E484Q can cause enhancement of ACE2 binding, transmission and immune evasion (15, 16) . The Omicron variant has 35 nonsynonymous mutations in the spike protein and 22 mutations in other viral protein-encoding genes. Among the nonsynonymous mutations in the S protein, only 11 mutations have been described in the previous VOCs (17) and this likely leads to the markedly different phenotype of Omicron in terms of transmission and escape from prior immunity.

Numerous assays and strategies to rapidly detect VOCs have been described (18) (19) (20) (21) (22) (23) . We had previously described assays for the detection of Alpha using real-time reverse transcriptase PCR (rRT-PCR) assays that specifically targeted the N501Y mutation and the 69-70 in the spike gene (24) . In this study, we improve upon that original assay design by multiplexing the targets and optimizing it for the detection of Omicron. We also describe three additional rRT-PCR assays that target key single nucleotide polymorphisms (SNPs) found in the spike gene of the recognized VOCs. These assays were initially validated using samples with known lineage status. A further analysis was carried out where lineage designation based on these assays was compared with genome sequencing prospectively.

The implementation of these high throughput variant screening assays promptly after the identification of VOCs worldwide has helped in the identification of circulating genotypes and emerging trends to guide public health policies. 

Five assays incorporating 10 separate markers were designed during the time period of this study. A triplex assay incorporating previously reported primers and probes to detect N501Y, Δ69-70, and an E gene target was developed to test all samples for Alpha (the E gene target acted as a surrogate marker of viral load in a sample, as the VOC assays are reliable only for samples of sufficient viral load); this assay is referred to as the N501Y+ Δ69-70 + E gene (Alpha) assay (24, 25) . Two duplex assays to detect 242-244 WT + K417N and E484K + K417T were also devised to detect Beta and Gamma as a second step for samples that resulted N501Y positive but Δ69-70 negative by the N501Y + Δ69-70 + E gene (Alpha) assay. Finally, a duplex assay for B.1.617 (detecting L452R + P681 WT) was developed as another second step in the testing process for samples negative for N501Y, once this parent lineage was recognized as significant. Conversion of this assay into a triplex (L452R + P681 WT + E484Q) was undertaken when B.1.617.2 (Delta) was recognized as a VOC rather than B.1.617.

If the defined constellation of mutations was detected by the variant assays, the sample was reported as belonging to the corresponding specific lineage. If the complete set of defining mutations was not detected or if additional changes were noted, the results were reported as "presumptive variant" and genome sequencing was performed for lineage confirmation. With the arrival of the Omicron variant in November 2021, the N501Y + Δ69-70 + E gene (Alpha) assay was modified for the detection of this variant (hence referred to as the N501Y + Δ69-70 + E gene [Omicron] assay) and was run alongside the 242-244 WT + K417N assay to identify Omicron-positive samples (those that were positive for all five markers). The E484K + K417T assay was discontinued since neither Beta nor Gamma were circulating in Alberta by that time. The N501Y + Δ69-70 + E gene (Omicron) assay was implemented on December 13, 2021, subsequent to which only Omicron and Delta were reported as VOCs and samples with all other mutation patterns were subjected to genome sequencing. See Figure 2 for a summary of how the VOC assays were interpreted based on the evolution of VOCs.

All primers and probes used are summarized in Table 1 The reverse-transcription step was performed at 50°C for 5 minutes followed by incubation at 95°C for 20 seconds. Amplification included 45 cycles of denaturation at 95°C for 3 seconds, followed by annealing, extension and data acquisition at 60°C for 30 seconds on the 7500 Fast Real-Time PCR system (ABI).

Viral RNA from the different specimen types was extracted on one of two platforms according to Consensus genomes from data generated with ONT were compiled through the artic 1.1.3 pipeline (https://github.com/artic-network/fieldbioinformatics). The Illumina data was processed with the OICR fork (https://github.com/oicr-gsi/ncov2019-artic-nf) of the ncov2019-illumina-nf pipeline (https://github.com/connor-lab/ncov2019-artic-nf), this pipeline was further updated to use freebayes as the variant caller (https://github.com/jts/ncov2019-artic-nf). The quality of the sequencing runs was assessed ncov-qc (https://github.com/jts/ncov-tools); pangolin was used to assign lineages (https://www.nature.com/articles/s41564-020-0770-5) and nextclade was used to detect mutations 

Diagnostic characteristics of the VOC assays such as analytical sensitivity, analytical specificity, reproducibility and accuracy are summarized in Table 2 . The analytical sensitivities using quantified invitro RNA ranged from 2 to 116 copies/reaction based on probit analysis. The assays did not react nonspecifically with other pathogens included in the specificity panel, demonstrating 100% analytical specificity. The %CV representing assay variability was calculated based on one high and one low viral load sample each for Alpha, Beta, Gamma, Delta and Omicron positives tested in triplicate on three independent runs. Variability calculations for the assays targeting each of the mutations show that the intra-assay variability ranges from 0.03 to 3.93% and the inter-assay variability ranges from 0.21 to 3.86%. Table 2 outlines the analytical sensitivity and specificity with their corresponding confidence intervals for the panels of samples described in the Materials and Methods. Sensitivity and specificity for the 242-44WT + K417N, L452R + P681WT + E484Q, and 69-70 + N501Y + E-gene (Omicron) assays were 100%. The sensitivity and specificity for the E484K + K417T was 100% and 98.57% respectively, one sample that was positive for the Gamma lineage by the TaqMan assay was assigned to the B. 

Results of the VOC assays were compared to the genome designation based on genome sequencing, only samples with greater than 85% genome coverage were used in order to avoid samples with incorrect lineage designation as a result of lower genome coverage. A total of 9494 samples were compared for Alpha, 3730 for Gamma, 3730 for Beta, 3349 for Delta, and 193 for Omicron. Table 3 includes the total number of positive and negative samples compared and the performance of the VOC assays in terms of clinical sensitivity and specificity including the 95% confidence intervals; also included are details on the mutations encountered in the oligonucleotide binding regions that resulted in false negative results.

The timeline for the implementation of the screening assays for the different VOCs is shown in Table 1 . 

Variants of concern pose a threat to the population because these strains can change the epidemiology in terms of transmission, severity of disease, vaccine efficacy and treatment options.

(angiotensin-converting enzyme 2) receptor and the targets for neutralizing antibodies. These mutations can also change the conformational B-cell epitopes leading to potential reductions in vaccine efficacy (29) . There is evidence that all VOCs are more transmissible than the wild-type virus (30) (31) (32) . A meta-analysis conducted on studies from June 1, 2020 to October 15, 2021 showed that Alpha, Beta, Gamma, and Delta cause more severe disease than the wild-type virus in terms of hospitalization, intensive care unit (ICU) admission, and mortality, and Beta and Delta carried a higher risk than Alpha and Gamma (33) . Study of a large cohort in Ontario, Canada, showed that compared with the wild-type SARS-CoV-2 strains, the adjusted elevation in risk for hospitalization, ICU admission and death was higher for Alpha, Beta and Gamma, and the increased risk with Delta was even more pronounced (34) .

Public health labs have needed to respond to the VOCs in a manner similar to the original SARS-CoV-2 by having the ability to detect and differentiate the VOCs in a high-throughput fashion with a quick turn-around-time. In response to these evolving variants, rRT-PCR assays targeting constellations of mutations characteristic for a particular VOC were designed, validated, and implemented in our J o u r n a l P r e -p r o o f jurisdiction for testing high volumes of specimens that were positive for SARS-CoV-2. The VOC testing and reporting algorithm evolved as the number of cases and prevalence of different VOCs fluctuated in our population. When the number of cases was low, all positive samples were tested for VOCs; however, when the cases were high, laboratories were under intense pressure to focus on performing high volumes of diagnostic COVID-19 testing and thus VOC testing was narrowed to only certain populations where it was deemed to be of a higher impact. This real-time surveillance of VOCs was used by public health personnel to control the spread of the virus in multiple settings, such as outbreaks in schools, congregate living facilities, and hospitals. After the initial detection of VOCs by the rRT-PCR assays, a proportion of the circulating VOCs and non-VOCs were further characterized by genome sequencing for surveillance. This allowed the monitoring of VOC sub-lineages if present and a comparison of the sequences circulating in our community to the globally circulating lineages. This strategy was also designed to detect the arrival of new lineages by preferentially characterizing all the non-VOC lineages (as determined by the VOC assays) by genome sequencing. For example, once Delta became the predominant lineage, all the samples that tested as non-Delta were characterized by NGS and the same strategy is in current use with the dominance of Omicron.

Comparison of the lineage designation using the VOC assays to genome sequencing shows that the VOC assays provide reliable results but with a much faster-turn-around time with suitable clinical sensitivity and specificity. An additional benefit of rRT-PCR assays is that samples with lower viral load that cannot be characterized by genome sequencing can be successfully tested for a VOC. Full genome sequencing of a significant proportion of samples that are interpreted as non-VOCs by the VOC assays also allows the detection of nucleotide changes in the primer and probe binding regions that could result in false-negative results by the VOC assays. As outlined in the results, four samples showed the C21709T mutation in the Δ69-70 probe, two samples showed G23069T in the N501Y probe and two samples had G22918A and T22917A changes in the L452R probe leading to false-negative results. This J o u r n a l P r e -p r o o f combined use of rRT-PCR assays and genome sequencing represents a powerful approach whereby large numbers of samples can have their VOC status determined rapidly while simultaneously prioritizing samples for a more complete characterization when they may represent an emerging lineage of medical and public health importance. This approach also focuses genome sequencing resources on high-priority samples so that reagents and labour are not needlessly used to sequence samples that can be genotyped using more routine rRT-PCR reagents and resources.

As reported in the literature, we noted limited transmission of the Beta lineage in Alberta, with increased spread of the Gamma lineage in comparison. The enhanced transmissibility of the Delta and Omicron lineages was evident with the sharp rise and near-complete dominance of the lineage in our population. Comparison of the five VOCs with respect to the reproductive number and growth rate from ten countries with the highest number of analyzed sequences for the five VOCs showed that the transmissibility was highest for the omicron variant followed by Delta, Alpha, Gamma and Beta respectively. The highest estimated growth rates and reproduction numbers were due to the Omicron variant indicating the highest transmissibility (35) . Data from another Canadian province of Ontario indicates similar trends in the rise of VOCs, after the initial detection of B.1.17 in Dec 2020, and increase to more than 90% was noted (36) and an increase for Delta from 2.2% in early April, to 83% in late May was noted (37). Estimates of VOC prevalence between Mar1 to Nov 15, 2021 in Ontario show that 99.3% of COVID-19 were of the Delta variant, 0.2% of the cases were estimated to be Alpha and 0.2% were estimated to be Beta, Gamma or Mu (38).

The implementation of high throughput variant screening assays promptly after the identification of VOCs worldwide, has helped to guide public health policies. Information on circulating VOCs by the screening assays has helped to prioritize samples for genome sequencing in our population resulting in optimal use of genome sequencing resources and robust surveillance of circulating genotypes and emerging trends to monitor the evolution of SARS-CoV-2 in the province. Thus the J o u r n a l P r e -p r o o f provincial strategy adopted was a combined approach to rapidly detect known VOCs while continuously monitoring for evolving mutations.

We are indebted to the ProvLab research and testing staff, as well as the clinical testing laboratories of Alberta Precision Laboratories and DynaLIFE Medical Laboratories for sending samples to ProvLab for variant testing. We would like to thank CanCOGeN for funding the sequencing reagents and PHAC for supplying reagents. Table 1 . Primers and probes for the detection of the Δ69-70 deletion, N501Y, K417T, K417N, E484K , 242-244 WT, L452R, E484Q and P681 WT mutations in variants of concern for SARS-CoV-2.

*Primers and probes for N501Y were designed by Ontario Agency for Health Protection and Promotion [PHOL, Ontario, Canada, (39)], the E484K assay was developed at PHOL by Alireza Eshaghi (publication pending) and shared with Alberta Precision Laboratories. All other primers and probes were deigned inhouse. Table 2 . Performance of the SARS-CoV-2 variant assays. *Analytical sensitivity refers to 95% limit of detection based on probit analysis using in-vitro transcribed RNA. Numbers for copies/reaction are rounded up to the next whole number. **Analytical specificity was based on cross-reactivity to 34 commonly found respiratory pathogens. ***Numbers of positive and negative samples used for the calculation of accuracy are outlined in the text. Table 3 . Prospective comparison of genome sequencing with the VOC assays. *In this comparison, VOC assay results were categorized in the following ways: Alpha-positive samples were positive for Δ69-70 deletion and N501Y alone; Beta-positive samples were positive for N501Y, E484K, K417N and negative for 242-244 WT; Gamma-positive samples were positive for N501Y, E484K and K417T; Delta-positive samples were positive for L452R, P681 WT and negative for E484Q; and Omicron-positive samples were positive for Δ69-70, N501Y, and K417N. ** Four samples showed the C21709T mutation in the Δ69-70 probe and two samples showed G23069T in the N501Y probe binding leading to false-negative results. ***Sample initially tested negative for the N501Y target, but tested positive on repeat. ****Low viral load sample with an E gene Ct= 30.45, E484K=34.16 and negative for K417T. *****Mutation in probe binding region at G22918A and T22917A causing failure of L452R assay. NED/CTAGCCATCCTTACTGCG/MGB-NFQ *Primers and probes for N501Y were designed by Ontario Agency for Health Protection and Promotion [PHOL, Ontario, Canada, (39)], the E484K assay was developed at PHOL by Alireza Eshaghi (publication pending) and shared with Alberta Precision Laboratories. All other primers and probes were deigned inhouse. 

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100% (97.32% to 100.00%) *Analytical sensitivity refers to 95% limit of detection based on probit analysis using in-vitro transcribed RNA. Numbers for copies/reaction are rounded up to the next whole number. **Analytical specificity was based on cross-reactivity to 34 commonly found respiratory pathogens. ***Numbers of positive and negative samples used for the calculation of accuracy are outlined in the text. 

The authors do not have any financial or personal conflicts of interest to declare.

 Five real-time reverse transcriptase PCR assays were designed to detect variants of concern. The targets included Δ69-70, N501Y, K417N, Δ242-244, K417T, E484K, L452R, P681 and E484Q. This has provided close to real-time molecular surveillance for variants or concern J o u r n a l P r e -p r o o f