key: cord-0866241-qug5hd2w
authors: Trujillo, M.; Cheung, K.; Gao, A.; Hoxie, I.; Kannoly, S.; Kubota, N.; San, K. M.; Smith, D. S.; Dennehy, J. J.
title: Protocol for Safe, Affordable, and Reproducible Isolation and Quantitation of SARS-CoV-2 RNA from Wastewater
date: 2021-02-17
journal: nan
DOI: 10.1101/2021.02.16.21251787
sha: 80ae9ffff6dfd4bb4ff2b7a3443b65c7340351ca
doc_id: 866241
cord_uid: qug5hd2w

The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Furthermore, the RNA obtained using this protocol, minus the pasteurization step, can be sequenced both using a targeted approach sequencing specific regions or the whole genome. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to support their efforts in monitoring SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. Owing to a pasteurization step, it is safe for use in a BSL1+ facility. This step increases the genetic signal of the virus while making the protocol safe for the personnel involved. This protocol could be used to isolate a variety of other clinically relevant viruses from wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.

The tracking of SARS-CoV-2 infections has most often involved the detection of SARS-38

CoV-2 RNA via RT-qPCR in biological samples obtained from patients that develop some of the 39 symptoms associated to COVID-19 [1] . One of the disadvantages of this approach is that if 40 much of the transmission within a population is asymptomatic or unsampled, infections from 41 these individuals may be overlooked [2, 3] . Additionally, SARS-CoV-2 sequencing efforts, while 42 occurring at a much faster rate and larger, more global scale than in previous pandemics, suffer 43 biases because genomic information is often obtained from seriously ill patients, but not from 44 patients who do not seek medical attention, which include asymptomatic patients, and those 45 with mild symptoms who choose to follow the CDC's advice and convalesce at home. If most 46 transmission within a population is asymptomatic or unsampled, genomes from these 47 individuals are expected to represent most of the viral population circulating within the 48 community. Recently the discovery of novel variants of concern in different regions of the world 49 has added another challenge [4, 5] , which is to monitor the proportion of individuals that carry a 50 particular variant in a geographical area. Given that SARS-CoV-2 has been detected in fecal 51 samples [6, 7] , and subsequently in wastewater [3, 8, 9] , wastewater is being tested in cities 52 around the world to determine SARS-CoV-2 prevalence in communities [10] [11] [12] . Furthermore, 53 isolation of SARS-CoV-2 RNA from wastewater coupled with high-throughput deep sequencing 54 provides an almost unlimited source of unbiased viral sequences, which can be used to monitor 55 frequencies of variants of concern in populations. 56

With the goal of sequencing SARS-CoV-2 RNA from wastewater, we developed a 57 protocol to extract and quantify viral RNA. The initial step in the development of this protocol 58 was the decision to pasteurize our samples at 60 for an hour on arrival at the laboratory. 59

Given that SARS-CoV-2 is a biosafety level 3 (BSL3) agent, inactivation of the virus before 60 processing is often required before samples can be processed in BSL2+ or BSL1+ laboratories. 61

Happily, as we report here, pasteurization did not impair our ability to detect SARS-CoV-2, but 62 . CC-BY-NC 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 February 17, 2021. ; https://doi.org/10.1101/2021.02.16.21251787 doi: medRxiv preprint 4 wastewater, we explored the effect of longer incubation times on viral RNA recovery. Longer 88 storage in PEG/NaCl led to slightly greater recovery. 89

As we were mindful of the need to find cost effective solutions, we investigated 90 alternative, kit-free approaches to RNA isolation. In our hands, TRIzol (ThermoFisher Inc.) 91 performed better than the QIAamp Viral RNA Mini kit (Qiagen Inc.). As TRIzol is cheaper per 92 sample than column-based kits, we adopted it for the final protocol. An added benefit of TRIzol 93 relevant to downstream sequencing applications is that TRIzol segregates RNA in a separate 94 layer from DNA, unlike column-based isolation kits, which isolate both RNA and DNA. 95

In addition to the RNA isolation method, we compared the performance of different RT-96 qPCR enzymes, TaqPath 1-Step RT-qPCR enzyme (Thermofisher Inc.) and One

Step 97

PrimeScript III enzyme (Takara Bio USA Inc. The RT enzyme from Takara was 25% cheaper 98 and had a similar performance to Taq-Path so we chose it for the final protocol. A broader 99 investigation of different enzymes may identify other satisfactory, cost-effective solutions. 100

Our protocol provides a reproducible and low-tech approach that allows the detection 101 and quantification of SARS-CoV-2. Pasteurization of the sample at the very beginning of the 102 protocol ensures the safety of the user. Preliminary results suggest that pasteurization may also 103 release the virus bound to the wastewater solids, enhancing recovery. Filtering and PEG/NaCl 104 concentration simplifies downstream processing. The extraction of RNA using TRIzol reduces 105 the cost significantly when compared to extraction column-style protocols using commercial kits. 106

We have been able to do both targeted and whole genome sequencing of the SARS-CoV-2 107 genome using this protocol but recommend removing the pasteurization step if this is the main 108 goal of the experiments. Table 3 . In addition, the Pecson et al. study offers strong 112 support for several of the primary claims of the present paper. First, the removal or non-removal 113 . CC-BY-NC 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 February 17, 2021. ; https://doi.org/10.1101/2021.02.16.21251787 doi: medRxiv preprint 5 of the wastewater solids did not show a clear systematic impact on outcomes. Second, 114 pasteurization resulted in a small, but significant, increase in recovery. Third, methodological 115 differences between teams had minimal impact on reproducibility and sensitivity, thus indicating 116 that our modifications to implement cheaper, simpler methods will not impair SARS-2-CoV-2 117 detection and quantification relative to other strategies. 118

We recognize that our protocol has some limitations. Our current protocol isolates the 119 RNA from 40 ml of wastewater and requires access to a centrifuge capable of reaching 12,000 x 120 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 February 17, 2021. ; https://doi.org/10.1101/2021.02.16.21251787 doi: medRxiv preprint 6 different times using different wastewater samples. Our purpose here is not to report regional 140 prevalence, but rather to demonstrate the reliability and consistency of our protocol. Key steps were optimized during the development phase of our protocol. Initially we 150 used Phi6 [14] as a spike in control. However, we found that Phi6 was rapidly degraded in the 151 pasteurization step of our protocol. Reports from the scientific community suggested that BCoV 152 would serve as a better control, however, we found that BCoV was significantly degraded by 153 pasteurization as well. Consequently, we switched to spiking samples with BCoV after 154 pasteurization and before the first centrifugation to remove solids. It would be interesting to 155 determine why BCoV was rapidly degraded by pasteurization, but an ostensibly similar virus, 156 SARS-CoV-2, was not. 157 . CC-BY-NC 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 February 17, 2021. ; https://doi.org/10.1101/2021.02.16.21251787 doi: medRxiv preprint 7 To ascertain the impact of pasteurization on SARS-CoV-2 quantitation, a single 158 California wastewater sample was divided into ten parts. Five of these replicate samples were 159 pasteurized and five were not. The positive impact of pasteurization on SARS-CoV-2 160 quantification is reflected in the increase of N1 copies/L of pasteurized versus unpasteurized 161 samples ( Fig. 2 ; paired t-test: t = 7.191, df = 4, p = 0.002). We speculate that incubation of 162 samples at 60 contributes to release of virus from wastewater solids. As an additional 163

advantage, pasteurization appears to increase repeatability of sample quantification. (ThermoFisher Inc.). TRIzol facilitates significantly better RNA recovery than the kit at a fraction 217 of the cost (Fig. 5 ; RM ANOVA: F= 1441, P < 0.0001). We note that we also found phenol-218 chloroform extraction to be less consistent than TRIzol on saliva samples, so while phenol-219 chloroform is likely even cheaper, we advise against its use in this protocol. TRIzol was 220 therefore chosen as the organic extraction method to compare with column approaches. 221

Importantly the supply of TRIzol is less impacted by supply chain issues. Additionally, TRIzol 222 removes DNA, but retains RNA, whereas column-based kits are unable to do so. If the intention 223 is to sequence RNA obtained from wastewater samples, TRIzol extraction provides a cleaner 224 sample with less contaminating DNA from non-SARS-CoV-2 genomes. As a caveat, because 225

TRIzol requires the careful extraction of an aqueous layer from a multilayered solution, TRIzol 226 extraction requires training and is best performed by experienced users. 227 . CC-BY-NC 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. In addition to comparing RNA isolation methods, we evaluated the performance of 237 different enzymes, including the TaqPath 1-Step RT-qPCR enzyme (ThermoFisher Inc.) and 238

One Step PrimeScript III enzyme (Takara Bio USA Inc.) Our results indicated that the One Step 239

PrimeScript III enzyme gave slightly better results (Fig. 6) . As the One Step PrimeScript III 240 enzyme was 25% cheaper and performed similarly to the ThermoFisher enzyme, we chose the 241 PrimeScript III enzyme for the final protocol. 242 . CC-BY-NC 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. preliminary results have shown that RNA extracted with our PEG/TRIzol protocol can be 256 sequenced using both traditional Sanger sequencing and NGS technology. However, we also 257 note that pasteurization reduced sequencing quality and output relative to unpasteurized 258 samples, so we recommend skipping this step if the intention is to sequence RNA obtained from 259 wastewater. 260 . CC-BY-NC 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) CC-BY-NC 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 February 17, 2021. ; https://doi.org/10.1101/2021.02.16.21251787 doi: medRxiv preprint

Detection of SARS-CoV-2 in Different 299 Types of Clinical Specimens

CoV-2 Transmission From People Without COVID-19 Symptoms

Tracking COVID-19 with wastewater

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor 308 plasma

Preliminary 310 RNA in the feces of COVID-19 patients

SARS-CoV-2 318 detection, viral load and infectivity over the course of an infection

SARS-CoV-2 RNA 322 monitoring in wastewater as a potential early warning system for COVID-19 transmission 323 in the community: A temporal case study. Science of The Total Environment

Implementation of environmental 326 surveillance for SARS-CoV-2 virus to support public health decisions: Opportunities and 327 challenges

Genome Sequencing of Sewage Detects Regionally Prevalent SARS-CoV-2 Variants

throughput sequencing of SARS-CoV-2 in wastewater provides insights into circulating 334 variants

USA, an ASU start-up company providing commercial services in 337 wastewater-based epidemiology. R.U.H. is the founder of OneWaterOneHealth, a non-338 profit project of the Arizona State University Foundation

Epub 2020/10/15

Saif LJ. Bovine respiratory coronavirus

Bacteriophage phi6: a Lipid-Containing Virus of 347

Pseudomonas phaseolicola

Detection of rotavirus A in sewage samples using multiplex qPCR and an evaluation of 352 the ultracentrifugation and adsorption-elution methods for virus concentration

Development and application of a one-step low cost procedure to concentrate viruses 357 from seawater samples

is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) CC-BY-NC 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 February 17, 2021. ; https://doi.org/10.1101/2021.02.16.21251787 doi: medRxiv preprint