key: cord-0863624-j1exeh1y
authors: Ahmed, Warish; Bivins, Aaron; Metcalfe, Suzanne; Smith, Wendy J.M.; Ziels, Ryan; Korajkic, Asja; McMinn, Brian; Graber, Tyson E.; Simpson, Stuart L.
title: RT-qPCR and ATOPlex sequencing for the sensitive detection of SARS-CoV-2 RNA for wastewater surveillance
date: 2022-05-16
journal: Water Res
DOI: 10.1016/j.watres.2022.118621
sha: 9079e8321f2b816c6c559201f0702813688ffb9e
doc_id: 863624
cord_uid: j1exeh1y

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to detect and quantify SARS-CoV-2 RNA in wastewater, while monitoring viral genome mutations requires separate approaches such as deep genomic sequencing. A high throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique has shown promise in detecting viral variants while also providing virus quantitation data. However, sensitivities of both RT-qPCR and sequencing analyses can be impacted through losses occurring during sample processing (e.g., sample handling, concentration, nucleic acid extraction and RT-qPCR), therefore process limit of detection (PLOD) assessments are needed to estimate the copy numbers of target molecule required to attain specific probability of detection. In this study, we compare the PLOD estimates of four commonly used RT-qPCR assays for quantification of SARS-CoV-2 (US CDC N1 and N2, China CDC N and ORF1ab) to that of ATOPlex sequence analyses through seeding known concentrations of gamma-irradiated SARS-CoV-2 into wastewater. Results suggest that among the RT-qPCR assays, US CDC N1 was the most sensitive, especially at lower SARS-CoV-2 seed levels. However, when results from all RT-qPCR assays were combined, it resulted in the greater detection rates than individual assays, suggesting that application of multiple assays is better suited for detection of trace levels of SARS-CoV-2. Furthermore, while ATOPlex offers promising approach to SARS-CoV-2 wastewater surveillance, this technology is appeared to be less sensitive compared to RT-qPCR and requires further refinements for routine monitoring and quantification of SARS-CoV-2. Nonetheless, the combination of both approaches (RT-qPCR and ATOPlex) may be a powerful tool to simultaneously quantify the viral load in wastewater and monitor emerging variants of concern.

Over the past two years, wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has grown to become a valuable tool for tracking coronavirus disease 2019 at the population level in many countries and regions (https://arcg.is/1aummW). Furthermore, many studies have described the potential of wastewater surveillance to provide early warning of COVID-19 in the community (Randazzo et Despite the high analytical and diagnostic sensitivities and specificities of these assays in the clinical context, their success in early detection of SARS-CoV-2 RNA circulating in community wastewater is mixed. The high dilution and fragmentation of viral RNA in wastewater pushes this technology to the limit and there is an ongoing need to improve method sensitivity and minimise false-negative results . RT-qPCR assay limitations include potentially reduced efficiency if mutations occur in the gene target region as was previously observed for assays targeting the S gene for the alpha variant in the UK (Grint et al., 2021) . Another limitation of RT-qPCR is low throughout, only one target (i.e., fragment of a genome) can be analysed at a time. To help overcome these limitations, many studies have used multiple RT-qPCR assays for the detection of SARS-CoV-2 RNA in wastewater (Ahmed et al., 2020a; Medema et al., 2020) , but requires increased analysis time. While the issue of time could be resolved by developing multiplex RT-qPCR assays, that requires additional and more complex method optimization, and the multiplex assay may not be as sensitive as a simplex assay (Parker et al., 2015) . The study also identified 57 unique mutations that were not present in the global database, that like the other studies indicates heterogeneity of SARS-CoV-2 variation in wastewater is greater in the clinic. This might reflect the presence of defective viral particles in feces and/or infections with novel virions. Along similar lines, Lin et al. (2021) applied targeted metagenomic sequencing of SARS-CoV-2 in wastewater and observed that the frequency and daily load of mutations associated with variants of concern (VoCs) were highly correlated with clinical incidence rates within the region of British Columbia, Canada. Based on these analyses, it is apparent that genomic sequencing of wastewater samples can be used to investigate the diversity of SARS-CoV-2 circulating in a community and identify new outbreaks.

Genomic sequencing of wastewater may not only shed light on the evolution of SARS-CoV-2 during an outbreak by identifying viral mutations, but it could also be applied as an approach for quantifying genomic fragments of SARS-CoV-2 as well. A recent study used a high-throughput sequencing platform (ATOPlex) that uses a multiplex tiled PCR-based enrichment technique and claimed that ATOPlex is capable of quantifying SARS-CoV-2 RNA in wastewater at concentrations that are at least one order of magnitude lower than RT-qPCR (Ni et al., 2021) . That proof-of-concept study compared the detection achieved by US CDC N1 and N2 RT-qPCR assays with ATOPlex, using a dilution series of cDNA samples generated from a commercially available SARS-CoV-2 RNA positive control, rather than seeding SARS-CoV-2 in wastewater samples. Therefore, the impacts of wastewater matrix interference on the ATOPlex assay limit of detection (ALOD) are not known.

Collectively, these studies demonstrate the potential application of sequencing approaches for monitoring the presence and allelic frequencies of SARS-CoV-2 RNA in wastewater. Along with clinical data wastewater-based metagenome sequencing can potentially identify emerging variants/lineages of clinical importance within a population. However, benchmarking RT-qPCR and sequencing approaches has not yet been performed and is required to understand and quantify the sensitivities and of these two detection methods.

The process limit of detection (PLOD) represents the analytical sensitivity of a sampling method after incorporating the efficiency of all the processing steps (e.g., sample handling, concentration, nucleic acid extraction, and PCR assays). The PLOD estimates the copy number of a target molecule required in the wastewater sample matrix to achieve a specific probability of detection (Ahmed et al., 2022 under review) . The primary objective of this study was to evaluate the PLOD of four RT-qPCR assays (US CDC N1 and N2, China CDC N and ORF1ab (CCDC N and CCDC ORF1ab) and

ATOPlex sequencing for the detection of SARS-CoV-2 RNA in wastewater. This was achieved by seeding a dilution series of known concentrations of gamma-irradiated SARS-CoV-2 virions into wastewater followed by primary concentration, nucleic acid extraction, and RT-qPCR and ATOPlex sequencing analysis. To the best of our knowledge, this is the first study to assess the SARS-CoV-2 PLOD for wastewater and provides important insights on the analytical limitations for trace detection of SARS-CoV-2 RNA in wastewater using RT-qPCR and ATOPlex sequencing.

Gamma-irradiated SARS-CoV-2 stock used in this study was kindly provided by our colleagues from the Australian Centre for Disease Preparedness, CSIRO. Gamma radiation process to minimize the potential risk associated with handling SARS-CoV-2 during experiments has been reported in our previous study (Ahmed et al., 2022) . The concentration of the SARS-CoV-2 stock was determined from three aliquots of the stock suspension using the CDC N1 RT-dPCR assay, as described elsewhere (Ahmed et al., 2022) . The concentration determined to be 4.60 ± 2.50 × 10 6 GC/µL Immediately prior to seeding experiments, (Ahmed et al., 2022) . 

Two trials (A and B) were conducted to determine the detection sensitivity of SARS-CoV-2 in wastewater samples by RT-qPCR and ATOPlex sequencing workflows. A dilution series with varying concentrations of gamma-irradiated SARS-CoV-2 were seeded into wastewater. The dilution series had 10-fold decrements and were prepared by serial diluting the stock suspension using DNase and RNase free water, and then seeding these serial dilutions into 50-mL wastewater samples. For the trials A and B, the seeded SARS-CoV-2 concentrations ranged from ~2.32 × 10 5 to 2.32 × 10 2 GC/50 mL and ~1.79 × 10 5 to 1.79 × 10 2 GC/50 mL, respectively along a serial dilution in 10-fold decrements.

Adsorption extraction (AE) method was used to concentrate SARS-CoV-2 from wastewater samples In this study, the read processing was carried out by following the SARS- 

To minimize RT-qPCR contamination, nucleic acid extraction and RT-qPCR set up were performed in separate laboratories. A sample negative control was included during the concentration process. An extraction negative control was also included during nucleic acid extraction to account for any contamination during extraction. All sample and extraction negative controls were negative for the analyzed targets. (Fisher, 1922) .

The RT-qPCR standard curves prepared from gamma-irradiated SARS-CoV-2 had a linear dynamic range from 6 × 10 5 to 6 GC/reaction (1. In Trial B at 10 -3 dilution detection rates of the US CDC N1 (55.5%) and CCDC ORF1ab (66.6%) assays outperformed US CDC N2 (22.2%) and CCDC N1 (11.1%) assays. An increased detection rate (77.7%) was observed when results from all RT-qPCR assays were combined in comparison to results from any single RT-qPCR assay (which ranged from 11.1 to 55.5%). ATOPlex sequencing produced three positives of nine seeded wastewater samples, and the detection rate (33.3%) was greater than the US CDC N2 (22.2%) and CCDC N1 (11.1%) assays but lower than US CDC N1 and CCDC ORF1ab (both 55.5%).

Between Trials A and B, the US CDC N1 assay detection rates were relatively consistent, while the US CDC N2 and CCDC ORF1ab detection rates were greater for Trial B than A, and the CCDC N1 detection rate was greater for Trial A than B. Fisher's exact test (Table 4) Most of these studies were conducted in countries with high COVID-19 prevalence such as USA, Canada, Belgium, France, and Netherlands. However, analysis of wastewater-based SARS-CoV-2 detection sensitivities between sequencing (i.e., targeting multiple genomic loci) and RT-qPCR (i.e., targeting a single genomic locus) has not been performed. In view of this, we compared diagnostic sensitivities from several RT-qPCR assays and ATOPlex amplicon-based sequencing by seeding serially-diluted gamma-irradiated SARS-CoV-2 in wastewater. We present detection results by RT-qPCR and ATOPlex sequencing workflows for scenarios when the seeded numbers of SARS-CoV-2 in wastewater samples are moderate to low (10 5 to 10 2 GC/50 mL).

While the data from this study allows a cross-comparison among RT-qPCR assays, however, making a direct comparison between RT-qPCR assays and ATOPlex sequencing is difficult due to several differences in processing and worflow; namely, the RT-qPCR assays used in this study are one-step (RT and PCR included in the same tube), while ATOPlex sequencing is a two-step multiplex PCR which amplifies the RNA target region in a single tube, and sequencing involved preparation of circularized single strand DNA from RNA. Another significant difference between these two strategies is that RT-qPCR assays target a small fragment of the genome (~60 to 160 bp), while ATOPlex utilizes 259 primer sets along the SARS-CoV-2 genome with amplicon tiles ranging in size from 159-199 bp. There are also differences in input nucleic acid concentrations, kits, and in the designation of samples to positive or negative detections.

This study was carefully designed to include a number of wastewater samples to capture the inherent variations in the wastewater matrix, rather than using a bulk wastewater. Two trails of experiments were conducted to obtain confirmatory results. For SARS-CoV-2 concentrations, we used an adsorption-extraction method which is reported to be less variable for concentrating SARS- Among the RT-qPCR assays, the detection rate of US CDC N1 assay was greater than other assays, suggesting application of this assay may be advantageous when the level of SARS-CoV-2 is low or near the limit of detection in wastewater. However, combining results from multiple RT-qPCR assays produced a greater detection rate than the individual assays alone. Multiple assays, including US CDC N1, should be used for trace detections and to avoid potential false negative results due to mismatches in the primer target sequence from mutations. Interestingly, ATOPlex sequencing was not as sensitive as the US CDC N1 nor combined RT-qPCR assay results, despite its use of 259 multiplexed primer sets to amplify the SARS-CoV-2 genome and despite 2-fold greater RNA input in this study design.

A recent study has highlighted the potential application of ATOPlex sequencing for wastewater surveillance and reported that ATOPlex sequencing was capable of quantifying SARS-CoV-2 RNA at concentrations at least one order of magnitude lower than the detection limit of RT-qPCR (Ni et Here, we show that the PLOD of RT-qPCR for the detection of SARS-CoV-2 in wastewater was lower than that of ATOPlex. Therefore, for applications where positive/negative detection is critical,

and wastewater viral concentrations are low, RT-qPCR is the preferred method. There are additional benefits of RT-qPCR, such as its shorter turnaround time (within 4.5 h from concentration to results for RT-qPCR versus 48-60 h for ATOPlex), as well as its lower cost per sample. However, the application of multiplex tiling PCR based sequencing offers several critical advantages for wastewater surveillance, such as the ability to detect and monitor genomic variants (e.g., variants of concern/interest and novel/emerging variants) of concern. It could also be argued that sequencing provides more convincing detection of SARS-CoV-2 genomic fragments at low viral concentrations with verification via read mapping at multiple sites across the genome, as high Cq values in RT-qPCR can be difficult to discern between true detection from non-target amplification for non-specific RT-qPCR assays. This is especially an issue in the wastewater matrix which has greater chemical and nucleic acid complexity compared to human clinical specimens and thus more opportunity for offtarget amplification or spurious probe hydrolysis. Therefore, a strategy of frequent RT-qPCR testing (e.g., daily) complemented by periodic multiplex tiling PCR sequencing (e.g., weekly or fortnightly) may represent a powerful combination for monitoring SARS-CoV-2 concentrations and evolutionary dynamics throughout the COVID-19 pandemic.  The PLOD for RT-qPCR assays for SARS-CoV-2 detection in wastewater was lower than ATOPlex sequencing, however combination of both technologies could boost detections sensitivity while enabling identification of genomic variants as they arise.

The views expressed in this article are those of the author(s) and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency. The U.S. Environmental Protection Agency through the Office of Research and Development provided technical direction but did not collect, generate, evaluate, or use the environmental data described herein.

☐ X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Table 3 Proportion of samples positive for SARS-CoV-2 RNA in Trials A and B of wastewater seeded at four concentrations using four RT-qPCR assays and Trial A 2.32 × 10 5 GC 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 10 -1 Dilution 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 10 -2 Dilution 8/9 (88.9) 1/9 (11.1) 9/9 (100) 7/9 (77.8) 9/9 (100) 6/9 (66.6) 10 -3 Dilution 6/9 (66.6) 1/9 (11.1) 3/9 (33.3) 1/9 (11.1) 8/9 (88.9) 1/9 (11.1)

1.79 × 10 5 GC 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 10 -1 Dilution 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 9/9 (100) 8/9 (88.8) Table 4 Fisher's exact test p-values to compare the positivity rate of each RT-qPCR assay and all RT-qPCR assays combined with ATOPlex sequencing. Table 5 Mean Cq values of RT-qPCR positive wastewater samples at the lowest two dilutions (10 -2 and 10 -3 ) in 

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We thank CSIRO Land and Water for strategic funding to complete this research project. We thank Urban Utilities for providing untreated wastewater samples.

 Table 2 Genome depth, coverage, and mapping rates of known concentrations of SARS-CoV-2 seeded in wastewater in Trials A and B. Average and range values were generated from each batch of dilution (9 samples each) in Trials A and B.