key: cord-0844548-i4fddhwf authors: Islam, Golam; Gedge, Ashley; Lara-Jacobo, Linda; Kirkwood, Andrea; Simmons, Denina; Desaulniers, Jean-Paul title: Pasteurization, storage conditions and viral concentration methods influence RT-qPCR detection of SARS-CoV-2 RNA in wastewater date: 2022-01-25 journal: Sci Total Environ DOI: 10.1016/j.scitotenv.2022.153228 sha: a364b0e4ee5822ca19157fd36d6e285b6113c509 doc_id: 844548 cord_uid: i4fddhwf The COVID-19 pandemic presents many public health challenges including the tracking of infected individuals from local to regional scales. Wastewater surveillance of viral RNA has emerged as a complementary approach to track and monitor the presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a variety of communities of different land use and population size. In the present study, we investigate how five different parameters (pasteurization, storage temperature, storage time, polyethylene glycol (PEG) concentration, and pellet mass) affect the detection of the SARS-CoV-2 N gene and fecal abundance indicator pepper mild mottle virus (PMMoV) gene. Pre-treatment of 24-hour composite wastewater samples (n = 14) by pasteurization at 60 °C resulted in a significant reduction of total RNA concentration and copies of the SARS-CoV-2 N gene/L (paired Student's t-test, P < 0.05). Comparing the wastewater samples collected from 6 wastewater treatment plants (WWTPs) for a storage period of 7 and 14 days at 4 °C, −20 °C and −80 °C, demonstrated a decrease in SARS-CoV-2 N gene copies/L when samples were stored for 14 days at −20 °C. Polyethylene glycol-NaCl for purification and concentration of viral particles from the wastewater samples demonstrated that a short PEG incubation of 2 h during centrifugation at 4 °C was sufficient for the consistent detection of the SARS-CoV-2 N gene from a 30 mL sample volume. Combined, this paper presents method recommendations for developing a reliable, accurate, sensitive, and reproducible estimation of the SARS-CoV-2 virus in diverse domestic wastewater samples. The ongoing COVID-19 pandemic has had an immeasurable impact on society, as evidenced by the loss of 5.32 million lives globally in over 81 affected countries as of December 2021 (WHO https://covid19.who.int/). It is particularly challenging for low-and middle-income countries to conduct mass testing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in large populations due to several logistical challenges such as trained personnel, supply shortages, and reporting delays in reverse transcriptase quantitative polymerase chain reaction Wastewater based epidemiology (WBE) can provide a complementary surveillance system to measure the SARS-CoV-2 RNA in wastewater at the community level and may have the potential to be used as an early-detection system. As an example, in the Netherlands, SARS-CoV-2 RNA was detected in wastewater six days before the first clinical cases were reported (Whitney et al., 2021) . Each of these parameters may contribute to variability in the detection and measurement of viral RNA targets such as SARS-CoV-2 and the fecal-normalizing-control pepper mild mottle virus (PMMoV). Due to these factors, there is a need to compare the performance of different protocols focused on RNA concentration, storage, extraction, and detection of the SARS-CoV-2 virus in wastewater to develop improved methodology that can be used consistently and with a high level of sensitivity. The goal of the present study was to test some of the technical parameters and conditions that influence the measurement and detection of SARS-CoV-2 RNA and PMMoV viral particles To compare the effects of pasteurization on SARS-CoV-2 and PMMoV RNA detection, 14 raw influent wastewater composite samples collected in random from all wastewater treatment sites were split into duplicate aliquots in falcon tubes and placed into two groups. One group of 14 sample tubes was pasteurized by incubating at 60 °C in a pre-heated water bath for 1 hour (Pecson et al., 2021) . The pasteurized and unpasteurized samples (without heat treatment) were concentrated by using the same PEG processing method described above. To determine the effect of storage on viral RNA decay in the composite wastewater samples, duplicate samples collected from 4 different WWTPs and 2 upstream pumping stations in the region of Durham, were transferred into falcon tubes and stored in either a 4 °C refrigerator, a -20 °C or a -80 °C freezer for a period of 7 and 14 days (n=72). Frozen aliquots of samples were thawed over a 1-hour period in a room temperature water bath prior to processing using the PEG/NaCl ultracentrifugation and RNA extraction method, as previously described, for 7 and 14 days. To precipitate the SARS-CoV-2 and PMMoV viral particles, 12 wastewater samples were selected from 4 WWTPs and one pumping station and were split into duplicate tubes by the addition of 30 mL of each wastewater sample to 10 mL of 4X polyethylene glycol (PEG)/NaCl buffer (40 % w/v PEG 8000 and 1.5 M NaCl, pH=7.2) in Nalgene™ Oak Ridge High-Speed PPCO Centrifuge Tubes (Thermo-Scientific, Waltham, Massachusetts, USA) and vortexed briefly (Lewis and Metcalfe, 1988) . To determine the efficacy of PEG incubation time, one J o u r n a l P r e -p r o o f Journal Pre-proof group of duplicates were ultracentrifuged immediately after the addition of PEG/NaCl with no incubation period, and the other set of duplicates were incubated for 12 hours at 4 °C without agitation prior to the ultracentrifugation step. All 24 samples were centrifuged using a SORVALL RC 6+ Ultracentrifuge (ThermoFisher Scientific, MA, USA) at 12,000 x g for 2 hours at 4 °C (Wu et al., 2020). To help solidify the pellet, after discarding the supernatant, a second centrifugation step at 12,000 x g for 10 minutes followed. To determine extraction efficiencies for all wastewater samples, a viral surrogate control was carried out by adding 3 µL from a 1000 infectious units/mL of human coronavirus 229-E (HCoV-229E) viral standard stock (Chik et al., 2021) to the lysis buffer prior to RNA extraction. Recovery efficiency data was not included due to the significant loss of surrogate virus within the blank matrix (matrix), which severely affected the calculation of % Recovery of HCoV-229E and thus the estimated recovery of the target SARS-CoV-2. Total RNA was extracted from the concentrated wastewater pellets using the RNeasy® PowerMicrobiome® Kit (Cat # 26000-50. Qiagen, Germantown, MD) with the following alterations from the recommended protocol: 100 µL of phenol-chloroform-isoamyl alcohol (2005) were employed to target a region of the PMMoV strain S genomic sequence. In order to determine RNA extraction efficiencies, the HCoV-229E gene was quantified using primers/probe designed by Dr. Lilly Pang (Alberta Provincial Lab, University of Alberta). All probe/primers used in this study and their sequences can be found in Table 2 . amplification/detection occurred. The dynamic range of our linear standard curve was between 1 X 10 5 copies/uL to 1 X 10 1 copies/uL. All experimental data were tested for normality using a Shapiro-Wilk test, and logtransformed when tests of normality failed. All statistical analyses were conducted using For the PEG incubation experimental data, a student's paired t-test (α = 0.05) was also used to test for significant differences in SARS-CoV-2 and PMMoV gene copies/L between pooled samples with no incubation (control) and 12-hour incubation times (treatment) (n=12). Lastly, a least-squares linear regression (goodness of fit) was used to assess pellet mass (wet weight in mg) as a predictor variable of gene signal of N and PMMoV gene copies/L (n=120). The analysis of total RNA from pasteurized and unpasteurized wastewater samples demonstrated that significantly higher total RNA concentrations (ng/µL) were recovered from unpasteurized wastewater in comparison to the pasteurized samples (student's paired t-test, p<0.05) (Figure 1 ). Further investigation of the SARS-CoV-2 N gene copy numbers revealed that N gene copies/L were significantly higher in unpasteurized samples compared to pasteurized samples (student's paired t-test, p<0.05) (Figure 2A The stability of SARS-CoV-2 RNA in wastewater may be another potential contributing factor accounting for the loss of SARS-CoV-2 RNA signal. Comparing the SARS-CoV-2 N gene copy numbers of pooled sample data from all WWTP sites, we observed that storage temperature (Table 4A) . For the normalization marker PMMoV, it was found the gene copies were 2-3 orders of magnitude higher that SARS-CoV-2 and that storage temperature was not a significant factor affecting PMMoV gene copies/L concentrations (Table 3) . However, when analyzing time as a factor, there was a significant difference in PMMoV gene copies/L (F-Statistic: 26.377, P<0.001, Table 3 and Figure 4 ). PMMoV demonstrated an increase in gene copies/L at 7 days but a greater The storage of samples is critical when immediate processing cannot be achieved. Samples may need to be re-analyzed to ensure quality control assays are met or retroactively be  We conclude that pasteurization of wastewater samples negatively impacted the detection of SARS-CoV-2 by decreasing the total RNA concentration and SARS-CoV-2 N gene copies/L measured, but not PMMoV gene copies. J o u r n a l P r e -p r o o f Table 2 . Primers and Probes used in this study. 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World Health Organization Virological assessment of hospitalized patients with COVID-2019 A new coronavirus associated with human respiratory disease in China SARS-CoV-2 Titers in Wastewater Are Higher than Expected from Clinically Confirmed Cases. mSystems Comparison of Clinical Characteristics of Patients with Asymptomatic vs Symptomatic Coronavirus Disease RNA Viral Community in Human Feces: Prevalence of Plant Pathogenic Viruses The authors wish to acknowledge the help and assistance of Ontario Tech University, The Regional Municipality of Durham's Health and Works Departments, and all their employees involved in the project during this study. Their time, facilities, resources, and thoughts provided throughout the study helped the authors greatly. In addition, the authors would like to declare J o u r n a l P r e -p r o o f Table 4A . * Percent change in SARs-CoV-2 N gene viral signal at 4 • C, -20 • C, and -80 • C.