key: cord-1051208-6l5rr20o
authors: Erster, Oran; Mendelson, Ella; Kabat, Areej; Levy, Virginia; Mannasse, Batya; Assraf, Hadar; Azar, Roberto; Ali, Yaniv; Bucris, Efrat; Bar-Ilan, Dana; Mor, Orna; Elul, Michal; Mandelboim, Michal; Sofer, Danit; Fleishon, Shay; Zuckerman, Neta S.; Bar-Or, Itay
title: Specific Detection of SARS-CoV-2 Variants B.1.1.7 (Alpha) and B.1.617.2 (Delta) Using a One-Step Quantitative PCR Assay
date: 2022-03-14
journal: Microbiol Spectr
DOI: 10.1128/spectrum.02176-21
sha: 3fa45be6d6c1ea157de29573c833ff4ba5c0b6b2
doc_id: 1051208
cord_uid: 6l5rr20o

In this report, we describe the development of a reverse transcription-quantitative PCR (RT-qPCR) assay, termed Alpha-Delta assay, which can detect all severe acute respiratory syndrome coronavirus 2 (SC-2) variants and distinguish between the Alpha (B.1.1.7) and Delta (B.1.617.2) variants. The Alpha- and Delta-specific reactions in the assay target mutations that are strongly linked to the target variant. The Alpha reaction targets the D3L substitution in the N gene, and the Delta reaction targets the spike gene 156 to 158 mutations. Additionally, we describe a second Delta-specific assay that we use as a confirmatory test for the Alpha-Delta assay that targets the 119 to 120 deletion in the Orf8 gene. Both reactions have similar sensitivities of 15 to 25 copies per reaction, similar to the sensitivity of commercial SC-2 detection tests. The Alpha-Delta assay and the Orf8(119del) assay were successfully used to classify clinical samples that were subsequently analyzed by whole-genome sequencing. Lastly, the capability of the Alpha-Delta assay and Orf8(119del) assay to identify correctly the presence of Delta RNA in wastewater samples was demonstrated. This study provides a rapid, sensitive, and cost-effective tool for detecting and classifying two worldwide dominant SC-2 variants. It also highlights the importance of a timely diagnostic response to the emergence of new SC-2 variants with significant consequences on global health. IMPORTANCE The new assays described herein enable rapid, straightforward, and cost-effective detection of severe acute respiratory syndrome coronavirus 2 (SC-2) with immediate classification of the examined sample as Alpha, Delta, non-Alpha, or non-Delta variant. This is highly important for two main reasons: (i) it provides the scientific and medical community with a novel diagnostic tool to rapidly detect and classify any SC-2 sample of interest as Alpha, Delta, or none and can be applied to both clinical and environmental samples, and (ii) it demonstrates how to respond to the emergence of new variants of concern by developing a variant-specific assay. Such assays should improve our preparedness and adjust the diagnostic capacity to serve clinical, epidemiological, and research needs.

markedly increased infectivity, which led to recurring outbreaks and a "take-over" effect of specific variants, such as B.1.1.7 (Alpha), in a matter of 4 to 5 weeks (1) . Other variants, such as B.1.351 (Beta) and P1 (Gamma), spread rapidly and showed a significant potential to become dominant (4, 5) but were eventually pushed aside by others. Variant B.1.617, initially identified in India in late 2020, spread rapidly, and one of its sublineages (B.1.617-2) became a globally dominant variant within a few months. The transmission pattern of Alpha and Delta variants suggests that they both spread from their country of origin, rapidly reaching North and South America, Europe, Africa, and Australia (4, 5) . In Israel, two large morbidity waves were associated with the introduction of new variants. The first wave was the Alpha variant high-morbidity wave, which started in mid-December 2020 and declined by mid-March 2021 following the successful nation-wide immunization campaign with two doses of the BNT162-b2 vaccine (6) . The current high-morbidity wave dominated by the Delta variant followed, which started in July 2021 in parallel to vaccine waning immunity and continues (7) . Immunological studies demonstrated that some of the emerging variants have the potential to evade, at least partially, the neutralizing effect of both convalescent patients and vaccinated individuals (2, 3, (7) (8) (9) . The spreading of such variants may therefore compromise the effectiveness of global vaccination efforts aimed to contain the COVID-19 pandemic.

A fundamental pillar of the campaign against COVID-19 is the ability to detect the presence and identity of SC-2 RNA in both infected individuals and environmental samples. This pressing need led to an unprecedented number of commercial detection kits based on molecular and serological principles, with various degrees of sensitivity, throughput, reliability, and turnaround time (see references 10-12 and references therein). Due to their epidemiological and medical importance, the rapid identification of circulating variants becomes a central tool in the fight against the pandemic.

In addition to the diagnosis of clinical samples, the monitoring of SC-2 RNA in environmental samples, mostly, but not exclusively, wastewater, proved as an important tool (6, 13, 14) . Detection of SC-2 RNA in such samples is exceptionally challenging due to their very low abundance and the presence of PCR inhibitors, which render this approach even more difficult. Several reports describe the successful identification of SC-2 RNA in environmental samples, and some describe the classification of the examined samples using sequencing (6, 15, 16) . Classification of samples using sequencing requires sufficient quality and quantity, which are often not available with environmental samples. Moreover, this approach is currently much more expensive than PCRbased assays and cannot be scaled-up readily to facilitate rapid screening of a large number of samples efficiently.

In order to address the need for rapid classification of SC-2-positive samples, several manufacturers released sets of molecular tests that specifically detect key mutations that are associated with increased transmissibility or vaccine resistance (Seegene, Thermo Fisher, and Kogene Biotech). However, most of these mutations are common to multiple variants and can therefore not be used to assign a sample of interest to a certain lineage with a high degree of confidence using a single test. We recently developed quantitative PCR (qPCR)-based molecular tests that target mutations that are strongly associated with variants Alpha and Beta, thereby enabling rapid classification of such samples with high confidence. We demonstrated that the selected target mutations successfully reflect the lineage of the examined sample, as confirmed by Sanger and whole-genome sequencing (17) . In this report, we describe the development and utilization of two qPCR tests that target mutations that are, thus far, unique to variant B.1.617 (Delta lineage). We combined one of these reactions with the N D3L reaction that detects the Alpha variant and the inclusive E-sarbeco reaction (18) we described previously to a new selective multiplex assay. This assay can determine if the examined samples are of lineage Alpha, Delta, or neither. We show that the new multiplex assay, which we term "Alpha-Delta assay," is rapid, sensitive, and reliable, as confirmed by sequencing. Finally, we demonstrate the capability of the new assay to detect and classify SC-2 RNA extracted from environmental samples.

Design of the S 157del and Orf8 119del reactions. Alignment of genomes identified as the Delta lineage from the Global Initiative on Sharing All Influenza Data (GISAID) database (https://www.gisaid.org/) with SC-2 reference sequence NC_045512 showed that all Delta sequences contained a substitution and a deletion in nucleotide positions 22045 to 22050, which translates into amino acid E-to-G substitution in position 156 and deletions in positions 157 to 158 in the spike protein (Fig. 1A) . Accordingly, a specific reaction was designed to detect that deletion, denoted S 157del reaction hereafter, by using a probe that can only bind to the mutated sequence (21987 probe) (Fig. 1B) . In order to establish that the selected mutations were indeed unique to the Delta lineage, global analysis of SC-2 genomes was performed using the NextStrain website (https:// nextstrain.org/) using the deletion in positions 22045 to 22050 as a search term. The 

Microbiology Spectrum resulting dendrogram showed that the double deletion was indeed unique to the Delta lineage and was absent from other known lineages (Fig. S1A in the supplemental material). The Delta genome alignment showed a second unique deletion mapped to nucleotide positions 28248 to 28253 in reference sequence NC_045512 at the C-terminal end of Orf8, spanning amino acids 119 to 120 of the protein ( Fig. 2A) . Global genome analysis performed as described for the 22045-to-22050 deletion showed that the deletion at positions 28248 to 28253 was also unique for the Delta lineage (Fig. 2B) . A specific probe was designed to identify the deletion-containing sequence (28199). Secondary structure simulation suggested that the original probe sequence is likely to generate a strong stem-loop structure, thereby significantly reducing the reaction efficiency. In order to avoid this undesired outcome, a single G-to-T substitution was incorporated 

Microbiology Spectrum at position 10 ( Fig. S1 ). Two primers were then designed to amplify the deletion region and complete the reverse transcription-quantitative PCR (RT-qPCR) assay. The sequences of the Orf8 119-120del primers and probe are detailed in Table 1 . Sequencing of the Cterminal domain (CTD) of the Orf8 gene from the same four samples classified by the S 157del assay as Delta confirmed the presence of the Orf8 119 to 120 deletion, which is the target of the Orf8 119del reaction (Fig. 2B ). Additional confirmation for the accuracy of the two reactions was performed by sequencing four samples that were identified as Delta suspected. Each sample was sequenced in the S 157del region and the Orf8 119del region. As shown in Fig. S2 , all samples contained both deletions. Development and evaluation of the Alpha-Delta assay. In order to facilitate detection of SC-2 RNA regardless of the lineage and identify the Alpha or Delta lineages in a single test, the three SC-2-targeting reactions (i.e., E-sarbeco, N D3L , and S 157del ) were combined in one multiplex assay termed "Alpha-Delta assay." A control reaction detecting the human RNAse P gene was also included in the multiplex assay for an endogenous control, as was performed previously (17) . Analytical sensitivity of the Alpha-Delta assay was evaluated using serial dilutions of mixed RNA targets of the four reactions combined in the assay. Each dilution was examined as follows: dilutions down to 5 Â 10 3 copies/reaction were tested in ten replicates. Dilutions of 5 Â 10 2 to 5 Â 10 0 copies/reaction were run in 20 repeats (Table S2 ). In order to ensure that the calculated number of the in vitro transcribed target molecules (IVT standards) provides a reliable estimation of the actual viral genome copies, serial dilutions of commercial SC-2 RNA were tested together with the IVT standards in the E-sarbeco reaction. The combined calibration curve confirmed that the results obtained with the IVT standards are consistent with those obtained with the commercial SC-2 RNA standard (red circled data points, Fig. S3A ). The limit of detection (LOD) for the SC-2 targets was determined to be 12 copies/reaction for the E-sarbeco reaction, 50 copies for the N D3L reaction, and less than 10 copies for the S 157del reaction. The LOD for the human RNAse P reaction Alpha-Delta Novel qPCR Assay Microbiology Spectrum was 50 copies/reaction (Fig. S3 ). The Orf8 119del reaction was developed as a confirmatory assay for inconclusive Delta-suspected samples. This reaction was used in combination with the E-sarbeco reaction that served as an inclusive SC-2 control reaction to ensure the presence of the SC-2 RNA. The analytical LOD of that reaction was determined to be 24 copies/reaction (Fig. S3 ). The specificity of the Delta-specific reactions was tested using RNA extractions from lineages 19A/19B (Wuhan lineage), Alpha, Beta, and Gamma. As detailed in Table S3 , all lineages were negative for both the S 157del and Orf8 119del reactions. Analytical sensitivity of the multiplex reaction in wastewater was evaluated as described previously (19) , and the minimum detection values were close to those obtained with standard sampling material (19, Erster and Bar-Or, unpublished data). Analysis of the amplification curves of the different Delta assay variations showed that in some samples, mostly with a high RNA concentration (quantification cycle [C q ] # 22), a weak signal in the 6-carboxy-2,3,3,5,7,7-hexachlorofluorescein (HEX) channel was observed, presumably resulting from low-affinity N D3L reaction (Fig. 3D ). However, in these cases, the C q values of the E-sarbeco reaction and the S 157del reaction (in Delta samples) were always at least 10 cycles earlier than those of the N D3L reaction, thereby clearly indicating that the sample was not of the Alpha lineage. Both the S 157del and the Orf8 119del reactions were very specific and did not give a positive signal in non-Delta samples (Fig. 3) . In order to exclude a possible mutual effect of the four reactions combined together, serial dilutions of Delta RNA from cultured cells were tested, showing similar sensitivity of both the E-sarbeco and S 157del reactions, thus confirming the maintained sensitivity of the Alpha-Delta multiplex (Fig. S3 ). Examination of clinical samples using the Alpha-Delta assay followed by whole-genome sequencing (WGS) analysis. Between January and May 2021, the prevalent lineage in Israel was Alpha. The Delta lineage began spreading in the country during mid-June 2021. The Alpha-Delta assay was therefore used to classify clinical samples whose lineage attribution was unknown in order to enable rapid epidemiological investigations and to aid public health decision-making regarding WGS prioritization. This assay was then used as a routine test at the Israel Central Virology Laboratory. From June to August 2021, over 1,400 clinical samples were classified as Delta, Alpha, or neither using this assay. This classification was subsequently confirmed by Illumina WGS analysis. The details of 62 such samples selected randomly are listed in Tables 2 and 3. All samples classified by the Alpha-Delta qPCR assay as suspected Delta were either identified as such by the WGS analysis or Alpha-Delta Novel qPCR Assay Microbiology Spectrum classified as "no lineage" (Table 2 ). Those who were designated "no lineage" were mostly with C q values higher than 32 and were all with 70% or less sequencing coverage (Table 3) . These samples, which were classified as "Delta-suspected" by the Alpha-Delta assay, were positive for the Orf8 119 to 120 deletion, but the S 157del region was not fully sequenced. Comparison of the PCR results with the Illumina WGS analysis showed that of the 62 samples randomly selected, 19 were not classified by the WGS analysis but were positive for the Delta-specific deletions (the S 157del , Orf8 119del , or both) ( Table 2 ). Examination of the C q values obtained for the WGS nonclassified samples showed that two samples had values of 27 and 29 (14,957 and 14,968, respectively), and all others were higher than 30 ( Table 2 ). The sequence coverage of the unclassified samples ranged between 20.9% and 70% (Table 3 ). These data suggest that the positive identification of both the Orf8 119del and the S 157del mutations by the qPCR assay enable a more definite classification of these samples, even in the absence of a complete sequencing coverage. The assay also detected a small number of Alpha lineage samples (two in the samples presented in Tables 2 and 3) , where the N D3L mutation was detected by both the qPCR assay and the WGS analysis (Tables 2  and 3 ). Detection of the Alpha and Delta lineage in wastewater samples. The capability of the Alpha-Delta and confirmatory Orf8 119del assays to detect the presence of the Delta lineage RNA in sewage samples was evaluated using samples from different periods of the COVID-19 pandemic, from December 2020 to mid-June 2021. As detailed in Table 4 , the assay correctly identified the samples from 2020 as non-Alpha and non-Delta. Samples collected during the peak of the third morbidity wave in Israel were all identified as positive for the Alpha variant, and samples collected from July 2021 were all identified as positive for the Delta variant ( Table 4 ). The C q values of the N D3L and S 157del reactions, when positive, were comparable to those of the E-sarbeco reaction, demonstrating sufficient sensitivity of these reactions with RNA extraction of wastewater. These results indicated that the Alpha-Delta assay is sufficiently sensitive to detect the presence of SC-2 in wastewater and determine whether the Alpha or Delta lineages or both are circulating in the examined regions. In order to evaluate the capability of the ORF8 119del reaction to correctly detect the Delta linage RNA in wastewater samples, it was applied to samples that were previously identified as non-Alpha, non-Delta, or as Delta positive. Samples selected randomly from 2020 were all negative for the presence of the ORF8 119del mutation, while recent samples from July 2021 were all identified as positive for the mutation (Table 5) . Although the C q values obtained with the ORF8 119del reaction were 2 to 3 cycles higher than those of the S 157del reaction in this test, all samples were correctly identified. Collectively, these results show that both the Alpha-Delta multiplex assay and the confirmatory ORF8 119del test can be used for examination of identification of the Alpha and Delta lineages in wastewater samples.

The emergence of SC-2 variants that have the potential to affect both COVID-19 spread and vaccine evasion poses additional challenges for current intensive diagnostic efforts worldwide. The primary goal of diagnostic SC-2 tests was initially to detect the presence of SC-2 RNA, either in clinical or environmental samples. However, the need for rapid classification of SC-2 samples for their lineage, once a sample is determined as SC-2 positive, becomes essential when attempting to monitor emergence or incursion of new variants into a country, a geographic region, or a community. Surveillance through sequencing is currently the ultimate tool to identify the emergence of new and potentially important variants (20) . However, whole-genome sequencing (WGS) is expensive and time-consuming, requires exceptionally skilled personnel, and cannot be readily scaled-up for high-throughput testing compared to rapid molecular tests (16, 21) . In this dynamic situation of emerging variants, rapid, cost-effective, and high-throughput identification of circulating SC-2 variants is required.

We recently reported on the development and utilization of a rapid multiplex RT-qPCR assay that successfully classified SC-2 samples as Alpha, Beta, or neither. We demonstrated that it could be readily scaled-up to accommodate high-throughput testing (17) . Here, we describe a new assay that combines general detection of SC-2, together with specific classification of the two major variants Alpha and Delta. Previous reports of in-house-selective assays, as well as commercial variant detection kits, rely mostly on the identification of common mutations that are not specific to a particular variant (22) (23) (24) . Other variant of concern (VOC) detection approaches rely on multistep expensive procedures (25, 26) . Such assays are not suitable for rapid determination of the identity of an examined sample based on a single assay. Confirmation of the Alpha-Delta assay results by Sanger and Illumina sequencing reinforced its importance as a first-line rapid and reliable tool for classification of major VOCs, obviating the need for further examination. The identification of both Orf8 and spike gene Deltaspecific deletions in samples with insufficient sequencing coverage demonstrated the usefulness of the Alpha-Delta assay in determining the identity of "sequencing-resistant" samples. Such problems can result from low RNA quantity (as evident by the C q values detailed in Table 2 ) or other factors that inhibit the sequencing procedure. This is particularly important in classification of wastewater-derived samples, where the target RNA is a pool from multiple individuals, is diluted, and the samples contain chemical inhibitors. The Alpha-Delta assay successfully detected the presence of the Delta variant even in very low RNA concentration, thereby enabling sensitive monitoring of the circulation of this variant during its incursion into Israel. This assay was also used for screening international arrivals, which is particularly important when attempting to prevent the spreading of new variants. While other reported approaches to detect SC-2 variants in international travelers rely on lengthy multistep procedures (25, 26) , this assay is rapid (;70 min from extraction to answer) and can be implemented in most standard laboratories that perform routine PCR tests. This is especially advantageous when an accelerated epidemiological investigation or other actions need to be commenced and complete genomic analysis is not available. The Orf8 119del reaction was used in this study as a confirmatory assay, complementing the first-line Alpha-Delta assay. This is useful to confirm ambiguous results and to provide additional support for lineage classification when sequencing analysis is not available or insufficient. The S 157del and the Orf8 119del reactions showed similar performance in both clinical and wastewater samples and can therefore be used in the current situation where both deletions are unique for the Delta variant. However, if a new variant will emerge where one of these mutations is present, reassessment of the multiplex combination should be performed in order to adjust the reactions to the required assay specificity. The ability to assemble different reactions into a single multiplex assay is advantageous with respect to the intended use of the assay, as we show here. When the Beta variant was introduced in Israel, we used the combination of the Alpha Beta and inclusive E reactions to classify the examined samples. Upon introduction of the Delta variant, we modified the assay and added a confirmatory reaction to address 12689  28  29  31  12694  28  29  31  12698  30  31  32  12733  28  29  31  12737  26  27  30  12741  28  28  31  12745  29  30  32  12754  27  28  30  12762  30  30  32  12766  29  29  32  Env_128  31  29  32  Env_129  33  31  34  Env_130  31  30  32  Env_134  33  30  34  Env_138  32  29  31  Env_140  30  28  31  Env_143  31  30  33  Env_144  29  28  30  Env_145  30  29  31  Env_146  36  33  33  Env_152  35  30  33  Env_163  29  30  32  Env_168  32  29  32  Env_172  33  32  35  Env_173  30  31  34  Env_177  30  32  34  Env_186  30  31  34  Env_197  29  29  32  Env_198  30  30  33  S-12686  31  31  34 a Randomly selected wastewater samples that were previously tested with the Alpha-Delta assay were subsequently examined using the confirmatory Orf8 119del test, and the Cq values for each reaction are shown. b NA, no amplification.

the pressing need to identify the Delta and Alpha variants but also identify non-Alphaand non-Delta-positive samples, all in a single 80-min standard RT-qPCR assay. The implementation of such a self-developed method is limited to laboratories that are able to combine research with diagnostic routine rather than high-throughput diagnostic laboratories. Another limitation is the rapid pace in which novel variants are emerging, which, in some cases, may obviate the need to implement such selective assays in routine diagnostic work. The inability to predict which emerging variants will become widespread poses a major obstacle when having to decide which assays to develop. Nevertheless, this study demonstrates that in addition to implementation of a useful diagnostic tool, the approach of developing a tailor-made variant-specific qPCR assay can improve the SC-2 diagnostic capability in any qPCR-qualified laboratory. Moreover, it can complement variant classification when WGS analysis either fails or is not available. Such an assay can therefore be valuable for epidemiological studies, enabling a quick and affordable assessment of the circulation of major variants in a community or in a geographic region of interest. It is conceivable that the emergence of future SC-2 variants, and possibly other infectious viruses, will warrant further development of such diagnostic tools.

Ethics statement. The study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of the Sheba Medical Center (7045-20-SMC). Patient consent was waived as the study used remains of clinical samples, and the analysis used anonymous clinical data.

Preparation of clinical and environmental samples. Clinical samples were prepared during the laboratory diagnostic routine from nasopharyngeal swabs, as described before (17) . RNA was extracted using either the MagNA Pure 96 system (Roche) or the PPS MagLEAD system. The extractions were used for routine clinical diagnostics and were incubated at 280°C for long-term storage prior to the novel assay development. Environmental samples were processed as described before (6, 19) . Briefly, sewage samples were centrifuged and filtered before extraction with either Nuclisense EasyMag (bioMérieux) or EMag extraction systems. Immediately after extraction, all samples were preserved at 280°C until PCR testing was performed.

Cell culture. All SC-2 culturing experiments were performed under biosafety level 3 (BSL3) conditions according to the Sheba Medical Center biosafety guidelines. In order to culture positive SC-2 samples, the medium from the selected collection tubes was filtered using a 0.22-mm filter (Millipore) and used for inoculation of Vero E6 cells. The cells were allowed to reach 70% confluency and were then incubated for 1 h at 37°C with 300 mL of the filtered medium. After 1 h, the medium was removed, and the cells were incubated in modified Eagle medium (MEM-EAGLE) with 2% fetal calf serum (FCS). Cells were monitored daily for the presence of cytopathic effect (CPE). Upon CPE onset, the culture medium was collected, and viral RNA was extracted. Resulting RNA extractions were kept at 280°C until use.

Design of the B.1.671-specific RT-qPCR assays. Sequences classified as B.1.617 were obtained from the GISAID database (https://www.epicov.org/epi3/frontend #2d1c1e) and were analyzed by alignment with reference sequence NC_045512 to identify mutated regions that can be used for differential analysis. Uniqueness of the selected mutations was confirmed by performing global analysis for the selected mutations in the NextStrain website to identify other lineages with identical mutations (https://nextstrain.org/ncov/gisaid/global). Corresponding primers and probes that detect only the mutated sequences were designed for each region and examined in silico for secondary structure formation, specificity, and compatibility with qPCR assays using Geneious software and the NCBI BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The primers and probes used in this study are detailed in Table 1 .

RT-qPCR assay. MDX106 inhibitor-tolerant RT-qPCR mix was from Bioline. Primers and probes were either from Metabion or from Merk-Sigma Israel. Reaction components were assembled as described in Table 6 using the MDX106 inhibitor-tolerant mix. The reactions were run in a CFX-96 thermal cycler (Bio-Rad) using the following parameters: 45°C for 10 min 10 s, 45 cycles of 95°C for 2 min 20 s, 95°C for 4 s, and 60°C for 22 s. Fluorescence was recorded at 60°C at the end of each amplification cycle. Results were analyzed using the CFX Maestro software (Bio-Rad).

Sanger sequencing of culture-derived and clinical samples. The N-terminal domain (NTD) of the SC-2 spike gene and the C-terminal domain (CTD) of SC-2 Orf8 were amplified using the primer pairs detailed in Table 1 . Primer pair T7-21494 Fwd 1 Cov19 22475 was used for the spike region amplification, and primer pair pT7 27945 Fwd 1 28525 Rev was used for the Orf8 region amplification. PCR was performed using the PCRBIO 1-step go reverse transcription-PCR (RT-PCR) kit according to the manufacturer's instructions. The PCR products were resolved by 1.2% agarose gel electrophoresis to confirm adequate amplification. Each product was then used as the template for the dye labeling reaction using the BigDye kit according to the manufacturer's instructions. Sequencing was performed using the ABI 3500 genetic analyzer.

Generation of in vitro transcribed standard RNA. RNA standards of the reaction targets were designed and synthesized as described before (17) . The primers for the amplification of the S 157del and Orf8 119del reaction targets are detailed in Table 1 . RT-PCR was performed with Fwd primers containing the minimal T7 promoter sequence. Resulting PCR products were purified using the Macherey-Nagel NucleoSpin gel and PCR clean-up kit according to the manufacturer's instructions. The purified products were then used for in vitro transcription with the T7-Megascript kit according to the manufacturer's instructions (Thermo Fisher). The concentration of the resulting RNA was measured using a NanoDrop spectrophotometer. All reaction products were kept at 280°C until use.

Evaluation of analytical and clinical sensitivity of the Alpha-Delta assay. In order to facilitate rapid classification of the examined sample, four reactions were combined in a single assay, as detailed below. The inclusive E-sarbeco, which detects SC-2 RNA regardless of the variant (6-carboxyfluorescein [FAM] fluorophore), N D3L reaction, which detects the Alpha variant (HEX channel), S 157del reaction, which detects the Delta variant (Texas Red channel), and an endogenous control reaction for the human RNase P gene (Cy5 channel). The details of the E, N D3L , and RNase P reactions were described previously (17) . The combined multiplex was termed Alpha-Delta assay. The analytical sensitivity of the Alpha-Delta assay was evaluated using serial dilutions of the in vitro transcribed targets (IVT) of each reaction. All four targets were mixed together in an initial concentration of 10 24 ng/mL, and 10-fold dilutions were prepared in nuclease-free Tris-EDTA (TE) buffer, pH 7.5 (IDT). Validation of the IVT standards was performed by running the E-sarbeco reaction with IVT dilutions together with dilutions of a commercial SC-2 RNA standard (Vircell). The analytic sensitivity of wastewater samples was evaluated by spiking IVT pool dilutions into SC-2-negative wastewater extraction, as described previously (19) .

The clinical sensitivity was evaluated by serially diluting positive samples in an extraction of a SC-2-negative sample. Conversion of each in vitro transcribed RNA product from nanograms to copies was performed using the SciencePrimer website calculator (http://www .scienceprimer.com/) according to the RNA molecule size (Fig. S1 in the supplemental material). For target concentration of 500 copies or higher per reaction, 10 repeats were tested. For less than 500 copies per reaction, at least 20 repeats were tested for each target.

Collection and preparation of wastewater samples. Wastewater collection was based on composite automated sampling for 24 h located in different wastewater treatment plants. The samples were transferred under cold conditions to the lab and were processed within 24 h. Prior to further processing, culture-derived, inactivated Coronavirus OC43 strain (COV OC43) was added to the medium as described previously (6, 19) . This was then used as an internal control for the concentration and extraction stages using the OC43 qPCR assay described previously (27) . The concentration process uses 20 mL of raw sewage in duplicates. The raw sewage was centrifuged at 4,700 Â g for 5 min. The supernatant was transferred to a new tube containing 0.5 g MgCl 2 (0.26 M). The tube was gently shaken for 5 min and then filtered through 0.45-mm pore-size, 47-mm diameter electronegative mixed cellulose ester membranes (MCE) membranes (Merck Millipore Ltd.). The membrane was immediately transferred to a new tube containing 3 mL of lysis buffer (NucliSENS easyMAG). The tube was gently shaken to extract the trapped RNA virus. Total nucleic acids were extracted using the NucliSENS easyMAG system (bioMérieux, Marcyl'Etoile, France) according to the manufacturer's instructions. Extracted nucleic acids were eluted in 55 mL of elution buffer and stored at 270°C until sequencing. All samples were tested for the presence of COV OC43 to verify proper concentration and extraction and absence of significant PCR inhibitors.

Whole-genome sequencing. A COVID-seq kit was used for library preparation as per the manufacturer's instructions (Illumina). Library validation and mean fragment size were determined by Tapestation 4200 via a DNA HS D1000 kit (Agilent). Libraries were pooled, denatured, and diluted to 10 pM and sequenced on a NovaSeq system (Illumina).

Data availability. The nucleotide sequence data of the SARS-CoV-2 variants were deposited in GenBank under accession numbers OM766203 to OM766210.

Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 1 MB.

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We wish to acknowledge the members of the Central Virology Laboratory for their technical assistance and helpful discussion of the results obtained in this study.