key: cord-0304149-e2f2gcnz
authors: Fuzzen, M. L. M.; Harper, N. B.; Dhiyebi, H. A.; Srikanthan, N.; Hayat, S.; Peterson, S. W.; Yang, I.; Sun, J. X.; Edwards, E. A.; Giesy, J. P.; Mangat, C. S.; Graber, T. E.; Delatolla, R. E.; Servos, M. R.
title: Multiplex RT-qPCR assay (N200) to detect and estimate prevalence of multiple SARS-CoV-2 Variants of Concern in wastewater
date: 2022-04-13
journal: nan
DOI: 10.1101/2022.04.12.22273761
sha: 03f925360a6648ed654c67783d7517214f3e8628
doc_id: 304149
cord_uid: e2f2gcnz

Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named N200, focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS-CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non-mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS-CoV-2 and its VOCs.

corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by 23 use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A 24 multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including 25 Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and 26 validated. The probe-based multiplex assay, named "N200" focuses on amino acids 199-202, a 27 region of the N gene that contains several mutations that are associated with variants of SARS- 28 CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the 29 targeted mutations and worked equally well in single-and multi-plex modes. To estimate 30 prevalence of each VOC, the abundance of the targeted mutation was compared with a non-31 mutated region within the same amplified region. The N200 assay was applied to monitor 32 frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected 33 between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the 34 Delta variant along with the introduction and rapid dominance of the Omicron variant were 35 monitored in near real-time, as they occurred nearly simultaneously at all six locations. The 36 N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC 37 monitoring programs or replace more laborious assays currently being used to monitor SARS- 38 CoV-2 and its VOCs. 39 Introduction the gene, was applied for detection and quantification of relative proportion of the Alpha VOC (Graber 64 et al., 2021) . Those authors were able to effectively trace the increase of the proportion of Alpha variant 65 in wastewater of Ottawa, Canada, in almost real time. The trends in incidence of Alpha were similar to 66 that of the number of clinical cases in the community. These various studies demonstrate that WBS can 67 rapidly provide information on incidence and prevalence of SARS-CoV-2 to authorities with sensitivity 68 and lineage specificity. RT-qPCR assays are specific and can be deployed rapidly across existing WBS 69 networks. The challenges with using RT-qPCR assays in surveillance of VOCs are the amount of time 70 needed to develop and validate new assays each time a new VOC emerges as well as increasing the 71 number assays being conducted to continually monitor for various VOCs and VOIs. 72 The objective of this study was to develop a multiplex RT-qPCR assay that could be used to 73 simultaneously screen for several VOCs, as well as a marker of the total SARS-CoV-2 present in the 74 sample so that an estimation of the relative proportion of VOCs can be reliably determined. The N gene 75 region was selected for several reasons, including that this region is targeted by the N1 and N2 assays, 76 developed by the US CDC, which are commonly used for monitoring wastewater (Lu et al., 2020) . There 77 is evidence to suggest that assays developed for the N-region of the SARS-CoV-2 genome have greater 78 sensitivity for measuring various VOCs of SARS-CoV-2 in wastewater than assays targeting than does the 79 S- (Yaniv et al., 2021) or the envelope regions (Pérez-Cataluña et al., 2021) . The N-gene was selected as 80 the target region as the target of the assay since it is also rich in mutations unique to VOCs (Kiryanov et 81 al., 2022) . Specifically, the 121-basepair region in the N-gene open reading frame (ORF) (nucleotides 82 28,837 to 28,958) that includes single or multiple nucleotide variants for each VOC was selected (Table  83 1). According to GISAID data summarized by nextrain.org (Hadfield et al., 2018) , the nucleotide 28880-84 28882 (AA N:203) have one of the highest entropy levels in the SARS-CoV-2 genome, at 0.972, with 85 nucleotides 28883-28885 (AA N:204) also having high entropy level of 0.711 (nextstrain.org; accessed 86 April 1, 2022). The non-synonymous functional polymorphisms present in this region include R203M, 87 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 present in Delta, which increases the spread of the virus (Syed et al., 2021) ; R203K and G204R, (both 88 present in Alpha, Gamma Lambda, and Omicron, which are related to increased infectivity, fitness and 89 virulence (Johnson et al., 2021; Lee et al., 2021; Wu et al., 2021); and T205I, present in Beta and Mu 90 variants that were predicted to have reduced antigenicity and greater affinity for HLA-I (Antonio et al., 91 2021) . The presence of multiple mutations in a single region allowed for the design of a single amplicon, 92 multiple probe assay, that can distinguish among several VOCs. A Universal probe, that targets an area 93 of the amplicon without mutations allows for the quantification of the total SARS-CoV-2 signal, 94 regardless of which variants are present in wastewater. This allows for the measurement of the total 95 SARS-CoV-2 signal as well as providing estimations of the relative abundance of individual VOCs. 96

Prior to being incorporated into a wastewater surveillance program, the N200 assay was 97 validated using several synthetic standards as well as samples of wastewaters. As an example of the 98 utility of this assay, between November and January 2021, samples from six wastewater treatment 99 facilities in Ontario, Canada, were examined with the N200 assay. During this period the N200 assay was 100 utilized to test for the presence and increased frequency of the R203K/G204R mutation (present in the 101 Omicron VOC), while comparing directly to the decrease in frequency of the R203M mutation (present 102 in the Delta VOC), relative to a VOC-agnostic Universal marker, all within a single RT-qPCR reaction. 103 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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To select an appropriate RT-qPCR target for testing wastewater, mutations that were indicative 106 of each VOC, encompassing amino acids N:202-205 were identified. Mutations selected for each VOC 107

and their known prevalence are presented in Table 1 . The template sequence used for designs were 108 retrieved from accession numbers provided by TWIST Bioscience (South San Francisco, CA, USA) for 109 synthetic controls 14 (Alpha), 16 (Beta), 17 (Gamma) and 23 (Delta) and sequences were aligned using 110

MAFFT (Katoh et al., 2002) . 111 120 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2022. ; https://doi.org/10.1101/2022.04.12.22273761 doi: medRxiv preprint RRID:SCR_001363) with the "qPCR" parameter selected. Candidate primers that passed were 121 then screened against NCBI's non-redundant nucleotide database using BLAST (Altschul et al., 122 1990 ) and Primer- BLAST (Ye et al., 2012) to maximize mismatches with as much non-target 123 genetic material as possible (human, microbes, food, etc.). 124 Five probes were designed to be selective for each of the mutations (Table 1 , 2), as well 125 as a universal probe for the "Universal" probe complementary to a nearby, highly conserved 126 (bp 28891-28905, mutation rates <0.87% according to covidcg.org) region of the amplicon to 127 detect total SARS-CoV-2 (Table 2, Figure 1 ). Probes were designed with the mutated base(s) at 128 their center using PrimerExpress v3.0.1 with a GC content of 40-60%. BHQplus technology (LCG, 129 Biosearch Technologies, Inc., California, USA) was used to increase the Tm of each probe, to have 130 a predicted Tm of 69°C, which enabled the design of short probes approximately 15 nucleotides 131 long. Probes were then tested for hairpins, homodimers, and heterodimers. Candidate probes 132 were also screened for % identity to non-target sequences using NCBI's BLAST tool (Agarwala et 133 al., 2016) .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2022. ; with synthetic templates assays were tested with wastewater RNA extracts to ensure successful 145 amplification. Temperature gradients and primer/probe concentration gradients were 146 performed to determine the optimal assay conditions in singleplex. 147 Cross-reactivity of probes was assessed by use of synthetic RNA controls. Each probe, in 148 singleplex, was tested for reactivity with TWIST RNA controls 2, 14, 16, 17 and 23 at a 149 concentration of 500 copies per reaction. Specificity was also determined in a triplex assay 150 iteration (Universal, R203M, and R203K probes) by use of digital PCR (dPCR) at multiple 151 concentrations of TWIST RNA controls (see dPCR section for additional details). 152 The N200 assay was validated in multiplex by comparing the efficiency, R 2 and y-153 intercept of standard curves, as well as the estimated concentration and proportion of variants 154 tested in wastewater relative to singleplex. Sensitivity of the N200 assay with the Universal, 155 R203K, and R203M probes were determined by use of a 12-point standard curve (TWIST 156 synthetic controls) with 15-replicates each. The limit of detection (LOD) was defined as the 157 standard concentration at which >95% of the replicates were detected. This was performed by 158 two independent labs (at the University of Waterloo and the Canadian National Microbiology 159 Laboratory -Winnipeg) using the same suppliers of primers and probes and standards.

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The copyright holder for this preprint this version posted April 13, 2022. To concentrate RNA, 250 mL samples were well-mixed by inversion, 40 mL aliquots 170 poured into 50 mL conical tubes with 4 g PEG-8000 and 0.9 g NaCl, then mixed on an orbital 171 shaker at low speed at 4°C for 2 h before being stored at 4°C overnight (Wu et al., 2020) . 172 Samples were then centrifuged at 12,000 x g at 4°C for 90 minutes with no brake. The CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2022. together with CDC_N1 probe and primers (Lu et al., 2020) were used to determine absolute 198 copy number (cp) of RNA in the following SARS-CoV-2 genomic RNA standards: TWIST 199 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (1) 218 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2022. ; https://doi.org/10.1101/2022.04.12.22273761 doi: medRxiv preprint 220 The N200 assay performed well with all probes in singleplex. When tested with a serial 221 dilution of TWIST standards, the singleplex standard curves were linear, had an efficiency near 222 100%, and the slopes and y-intercepts were comparable between each of the probes (Table 3) . 223 The N200 assay performed similarly when tested in singleplex, duplex and triplex (Table 3 ). All 224 probes targeting mutations were found to be highly specific with little to no cross-reactivity for 225 non-target templates using qPCR (in singleplex; Table 4 ). Using RT-qPCR, this was demonstrated 226 by the lack of amplification on all VOC TWIST templates without the target mutation (Table 4 ).

Some cross-reactivity was observed between the T205I probe and the Wuhan variant control 228 (TWIST Control 2; Table 4 ), but this accounted for less than 5% of the signal observed on the 229 homologous template. Positive amplification with similar sensitivity was seen on of all TWIST 230 templates tested was observed with the Universal probe (Table 4 ). High target specificity of 231 each probe was confirmed by RT-dPCR, where in the presence of a high concentration of non-232 target template, almost no amplification was detected (Table 5 ). Copy numbers reported by the 233 mutation probes (R203M, R203K) were comparable to the copy numbers reported by the 234 Universal probe in the RT-dPCR reactions at multiple concentrations, which further suggests 235 that each probe has similar sensitivities and efficiencies ( Table 5 ). The LOD (95% detection) was 236 determined to be 4-6 copies per reaction for the Universal, R203K, and R203M probes 237 respectively when tested in two separate laboratories.

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The copyright holder for this preprint this version posted April 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Performance of N200 assay with samples of wastewater 250 The triplex N200 assay was applied to wastewater samples throughout 

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The copyright holder for this preprint this version posted April 13, 2022. In addition to R203M (Delta) and R203K/G204R (Omicron/Alpha) targets, probes 284 targeting the T205I (Beta) mutation and the silent mutation in the amino acid S202 (Gamma), 285 allow for surveillance of known VOCs using a single assay. Having multiplexed, variant specific 286 probes that are compared to a Universal reference within the same amplicon is beneficial for 287 improved consistency and accuracy of measuring relative prevalence of variants. As the gene 288 targets are all in the same 121 bp amplicon, there is minimal concern about the dynamics of 289 different regions of the SARS-CoV-2 genome in either the wastewater system itself, or in the 290 extraction process. Because the reactions occur together in a multiplexed assay there is 291 reduced variability and replication compared to running assays separately. This approach saves 292 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 resources and time, as fewer reactions are run per sample. Critically, this assay also saves 293 sample volume, which is often limited, allowing for additional assays to be run on the same 294 extract or reducing the need for multiple extractions to be performed. Ultimately it makes the 295 process more efficient, reduces turnaround time and improves quality. 296 The combined use of Universal and VOC-specific probes in the N200 assay to estimate 297 prevalence of VOCs in wastewater is advantageous for several reasons. First, since when 298 multiplexed, the mutation and comparator are being assessed within the same amplicon as well 299 the same reaction, this method provides increased precision of VOCs prevalence estimates. 300 Additional confidence in prevalence of VOCs is provided by the fact that the mutation and 301 Universal signals can be quantified using the same standard. In comparison, other VOC qPCR 302 assays estimate prevalence by comparing a mutant allele to a genetic marker of SARS-COV-2 in 303 another amplicon, /genetic locus such as the CDC_N1 (Vogels et al., 2021; Wolfe et al., 2021), 304 or by comparing the frequencies of the mutant and "wild-type" (e.g., ancestral/endemic allele) 305 at the same locus (Chan et al., 2022; Graber et al., 2021; Lee et al., 2021; Peterson et al., 2022) . 306 While these assays can produce accurate data (from both analytical and clinical perspectives), 307 there are additional controls needed to account for variations in efficiencies of variation (inter-308 assay variability and matching standard quantities), that are eliminated by the N200 assay. 309 Estimations of VOC prevalence are important during wastewater surveillance SARS-CoV-2 in 310 that they can provide useful information to public health units in a timely manner. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 13, 2022. ; https://doi.org/10.1101/2022.04.12.22273761 doi: medRxiv preprint first detected the R203K mutation in a sample collected from GE Booth (Peel Region) on 315 December 3 rd , 2021. While it is possible that this early detection of the R203K/G204R mutation 316 could have been a variant other than Omicron, within a week of this detection, the 317 R203K/G204R mutation was consistently detected in the wastewater at this site while R203M 318 decreased in proportion. The use of two mutation targets provided confidence in the data as it 319 was being collected and reported because both a decrease in the proportion of the R203M 320 mutation (Delta) and an increase in the proportion of the R203K/G204R mutations (Omicron) 321 occurred simultaneously. This data was reported to public health partners within a week of 322 samples being collected and this was immediately disseminated to the public by the Public 323 Health Unit as part of a weekly Epi-Report (https://data.peelregion.ca/documents/profile-of-324 covid-19-cases/explore). Rapid emergence of Omicron presented a major challenge for clinical 325 testing and sequencing capacity. This was also confounded by the holiday season in Ontario. 326 The wastewater data in general, quickly became important to Public Health Units as the clinical 327 testing became less reliable because of changes to testing eligibility in Ontario. They were able 328 to confirm the rapid replacement of Delta with the Omicron variant using wastewater that was 329 independent of clinical testing (Arts et al., 2022) . 330 The N200 multiplex qPCR was developed to improve estimates of the proportion of 331 various variants in wastewater, while also making the assay more efficient to use in terms of 332 reduced use of extracts, reagents, and time. The assay was validated using synthetic templates 333 and was successfully applied to monitor for the emergence of the Omicron variant in several 334 communities in Ontario. Although, this assay is one among several now available for detection 335 of VOCs in wastewater, the unique design, with a single amplicon, multiple target mutations, 336 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 13, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 and universal comparator targeting a highly mutable area of the N-gene make it very useful in 337 wastewater-based surveillance of SARS-CoV-2. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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