key: cord-0884544-eiyv9d4c
authors: Torii, Shotaro; Oishi, Wakana; Zhu, Yifan; Thakali, Ocean; Malla, Bikash; Yu, Zaizhi; Zhao, Bo; Arakawa, Chisato; Kitajima, Masaaki; Hata, Akihiko; Ihara, Masaru; Kyuwa, Shigeru; Sano, Daisuke; Haramoto, Eiji; Katayama, Hiroyuki
title: Comparison of five polyethylene glycol precipitation procedures for the RT-qPCR based recovery of murine hepatitis virus, bacteriophage phi6, and pepper mild mottle virus as a surrogate for SARS-CoV-2 from wastewater
date: 2021-10-03
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2021.150722
sha: b2f4c04f5bbe4d7bf249deffab539e239edc1d7e
doc_id: 884544
cord_uid: eiyv9d4c

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery of MHV (0.070%–2.6%) and phi6 (0.078%–0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (7.9 × 108 to 5.5 × 109) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation (r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

Development of sensitive methods for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater is a key imperative owing to the increasing attention on wastewater-based epidemiology (WBE) (Kitajima et al., 2020; Zhu et al., 2021) . Highly efficient methods for primary concentration and the downstream molecular processes, including nucleic acid extraction, reverse transcription (RT), and quantitative polymerase chain reaction (qPCR), are required.

Many researchers have recently proposed novel analytical methods (Ahmed et al., 2020a; Graham et al., 2020; Whitney et al., 2021) or compared the recovery efficiency of the various conventional methods (Chik et al., 2021; LaTurner et al., 2021; Pecson et al., 2021) .

Polyethylene glycol (PEG) precipitation is one of the methods for virus concentration from environmental water samples (Haramoto et al., 2018; Lewis and Metcalf, 1988) .

The polymer, PEG, preferentially traps solvent and sterically excludes proteins (e.g., virion) from the solvent phase. This allows for concentration of the proteins and their precipitation once their concentrations exceed the saturated solubility (Atha and Ingham, 1981 ; Lewis and Metcalf, 1988) . One of the advantages of PEG precipitation is that it can be performed with basic laboratory equipment (Ahmed et al., 2020b ) with a relatively low running cost compared to other methods (e.g., ultrafiltration). Several studies have reported its applicability for the detection of SARS-CoV-2 RNA in wastewater (Hata et al., 2021; Kumar et al., 2020; Torii et al., 2021; Wu et al., 2020) .

However, the procedures of PEG precipitation highly depended on implementers. For bovine serum (FBS) in a 75 cm 2 flask. Semi-confluent DBT cells were inoculated with MHV and incubated in MEM with 1% FBS at 37℃ (5% CO 2 ) for 3 days. Then, the flask was frozen and thawed once to recover MHV from the cells. The suspensions were centrifuged at 3,500 g for 15 min. The supernatant was filtered through a 0.2-μm cellulose acetate membrane (DISMIC-25CS, Advantec, Tokyo, Japan).

Bacteriophage phi6 (NBRC 105899, National Institute of Technology and Evaluation (NITE), Tokyo, Japan) was propagated using P. syringae (NBRC14084, NITE) as the host bacterium. To prepare the phi6 stock, P. syringae was propagated in Luria-Bertani broth at 28℃ for 6 h, subsequently inoculated with phi6, and incubated overnight. The suspensions were centrifuged at 3,500 g for 15 min. The supernatant was filtered through a 0.2-μm cellulose acetate membrane (Advantec).

MNV S7-PP3 strain was propagated on RAW.264.7 cells, as described elsewhere (Kitajima et al., 2008) . All propagated virus stocks were stored at 4℃ before the experiment.

A total of 34 raw wastewater samples were used in the experiment. Samples of raw wastewater (300 mL) were collected weekly from July 1 to October 19, 2020 (17 consecutive weeks) at wastewater treatment plants (WWTPs) A and B, both of which are in Kanto area in Japan, and stored at −20℃. Information pertaining to WWTPs A and B is provided in the supplemental information (SI) (see Table S1 ). The basic water quality parameters of raw wastewater at WWTP A were as J o u r n a l P r e -p r o o f

Each thawed raw wastewater sample was divided into five 41-mL aliquots. Each aliquot was spiked with 41 μL of propagated MHV and phi6 as whole process controls (WPCs) to obtain the final concentrations of 4.0 × 10 5 and 1.6 × 10 5 copies/mL, respectively. As a control, 41-mL MilliQ water (Millipore, Tokyo, Japan) spiked with the same amount of MHV, and phi6 was prepared in duplicate to determine the initial concentrations (see Figure S1 for the results). The spiked raw wastewater and MilliQ water were immediately placed in −20℃ freezer and stored overnight. Each set of samples was transported at <−70℃ to five laboratories and stored in a freezer at −20℃ for up to one month to perform each PEG precipitation procedure, RNA extraction, and the RT process.

The whole experimental scheme is illustrated in Figure 1 J o u r n a l P r e -p r o o f Figure 1 . Flow diagram of sample processing for the comparison of five PEG precipitation procedures. PEG precipitation, RNA extraction, and RT were performed in each laboratory. In the laboratory of the University of Tokyo, the spiked wastewater was also directly subjected to RNA extraction, RT, and qPCR for the measurement of the indigenous PMMoV concentration in the unconcentrated samples. All the runs of qPCR were performed at the laboratory of the University of Tokyo.

The raw wastewater sample was concentrated as described elsewhere (Hata et al., 2021; Torii et al., 2021) . A 40-mL aliquot of raw wastewater was centrifuged at 3,500 g for 5 min to remove the suspended solids. The supernatant was supplemented with 4.0 g of PEG8000 and 2.4 g of NaCl to obtain the final concentrations of 10% (w/v) and 1.0 M, respectively. The mixture was incubated overnight in a shaker at 4℃. Then, the J o u r n a l P r e -p r o o f Journal Pre-proof mixture was centrifuged at 10,000 g for 30 min. After carefully discarding the supernatant, the precipitate was resuspended with 10 mM phosphate buffer (0.5 mL).

The final volume of the concentrate was 0.68 ± 0.05 mL.

This procedure was identical to L.Long except for the incubation time. In this protocol, the incubation period was reduced to 2 h. The final volume of the concentrate was 0.62 ± 0.05 mL.

The raw wastewater sample was concentrated using the protocol recommended by the IDEXX Laboratories (Westbrook ME, USA) with slight modifications (https://www.idexx.com/files/sample-concentration-protocol-for-wastewater-surveillanc e.pdf). Briefly, a 40-mL aliquot of raw wastewater was centrifuged at 4,700 g for 30 min at 4℃ to remove the suspended solids. The supernatant was supplemented with 4 g of PEG8000 and 0.9 g of NaCl to obtain the final concentrations of 10% (w/v) and 0.4 M, respectively, followed by vortex mixing for 10 min. The mixture was directly subjected to centrifugation at 12,000 g for 100 min at 4℃ without incubation. The supernatant was discarded, leaving approximately 5 mL of the mixture in the tube. The mixture was subsequently centrifuged at 12,000 g for 5 min at 4℃. After carefully discarding the supernatant, the precipitate was resuspended with 600 µL of PCR-grade water, followed by vortex mixing and spin down. The final volume of the concentrate was 0.65 ± 0.17 mL.

A 40-mL aliquot of raw wastewater was filtered through a hydrophilic polytetrafluoroethylene membrane (Millipore) with a pore size of 0.2 μm. The filtrate J o u r n a l P r e -p r o o f Journal Pre-proof was supplemented with 4.0 g of PEG6000 and 0.9 g of NaCl to obtain the final concentrations of 10% (w/v) and 0.4 M, respectively. The mixture was incubated overnight in a shaker at 4℃. Then, the mixture was centrifuged at 12,000 g for 60 min.

After carefully discarding the supernatant, the precipitate was resuspended with 0.5 mL of TRIzol reagent (Thermo Fisher Scientific, MA, USA). The final volume of the concentrate was 0.66 ± 0.03 mL. Note that the TRIzol reagent was used as a suspension medium of PEG precipitates. The use of TRIzol reagent aimed to immediately lyse the protein in the precipitates, resulting in virus inactivation, which minimizes the microbial health risks for laboratory personnel. The suspensions were not treated by chloroform but were directly subjected to RNA extraction using QIAamp Viral RNA Mini Kit (see 2.4).

A 40-mL aliquot of raw wastewater was incubated at 60℃ for 90 min. The pretreated raw wastewater was supplemented with 3.2 g of PEG6000 and 0.9 g of NaCl to obtain the final concentrations of 8% (w/v) and 0.4 M, respectively. The mixture was incubated at 4℃ overnight in a shaker. Then, the mixture was centrifuged at 10,000 g for 30 min.

After carefully discarding the supernatant, the precipitate was resuspended with 0.5 mL of TRIzol reagent. The final volume of the concentrate was 1.14 ± 0.33 mL. The suspensions were not treated by chloroform but were directly subjected to RNA extraction using QIAamp Viral RNA Mini Kit (see 2.4).

four laboratories ( Figure 1 ).

Prior to RNA extraction, a 140-μL aliquot of each sample was seeded with 5-μL MNV as a molecular process control (MPC). Comparison of the MNV concentrations between the concentrates or unconcentrated wastewater samples and the spiked MilliQ water allows the evaluation of the total recovery efficiency of the molecular process (i.e., extraction-(RT-)qPCR efficiency) (Haramoto et al., 2018) . The samples were processed by QIAamp Viral RNA Mini Kit (QIAGEN, Tokyo, Japan) to obtain an RNA extract with a final volume of 60 μL. The RNA extract was subjected to RT on the same day in each laboratory.

Before the RT reaction, 8-μL subsample of RNA extract was incubated at 95℃ for 5 min, followed by 4℃ for 1 min to denature the double-stranded RNA. Then, 8-μL heat-incubated RNA and 35-μL non-incubated RNA were subjected to RT to synthesize cDNA. High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used according to the manufacturer's instructions. The obtained cDNA was stored at −20℃ at each laboratory and transported with ice packs to the laboratory of the University of Tokyo. The transported cDNA was stored at −20℃ until qPCR.

All qPCR assays were run with StepOnePlus TM Real-Time PCR System (Thermo Fisher Scientific) at the laboratory of the University of Tokyo. Quantification of MHV, phi6, PMMoV, and MNV was performed with TaqMan-based qPCR assays using previously reported primers and TaqMan (MGB) probe (Besselsen et al., 2002; Gendron et al., 2010; Haramoto et al., 2013; Kitajima et al., 2008; Zhang et al., 2006) .

SARS-CoV-2 RNA was detected with two TaqMan-based qPCR assays, CDC-N1 and CDC-N2, using the reported primers and probe (Centers for Disease Control and J o u r n a l P r e -p r o o f Journal Pre-proof Prevention, 2020) . The primers and probes used in the present study are listed in Table   S2 . The thermal cycle conditions are listed in Table S3 . Briefly, 5 μL of synthesized cDNA was mixed with 15 μL of reaction mixture containing forward primer, reverse primer, and TaqMan (MGB) probe and 10 μL of TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific). All reactions were tested with duplicated qPCR reactions. Negative control was included for every qPCR run.

A standard curve was generated every run from ten-fold serial dilutions of gBlocks for the assays of MHV, phi6, PMMoV, and MNV (Integrated DNA Technologies, Coralville, IA, USA) or plasmid DNA for the assays of CDC-N1 and CDC-N2 (Cat:

10006625, Integrated DNA Technologies) containing the target sequence (5 × 10 5 to 5 × 10 0 or 5 × 10 4 to 5 × 10 0 copies/reaction). The number of viral genome copies per qPCR reaction was determined by each standard curve. For CDC-N1 and CDCN-2 assays, the samples that showed Ct values lower than 40 for at least one out of the duplicated reactions were considered positive for SARS-CoV-2.

The slope, intercept, R 2 , and amplification efficiency of the standard curves of each assay are presented in Table S4 .

The whole process recovery efficiency (W) (Eq. (1)) and molecular process recovery efficiency (M) (Eq. (2)) are given below. Note that the W depends on the efficiency of the concentration process and the downstream molecular processes (i.e., RNA extraction, RT, and qPCR), while M depends only on the efficiency of the molecular process. The observed concentration of indigenous PMMoV, C obs_PMMoV (copies/L) was calculated using Eq. (3).

where C conc_PMMoV represents the concentration of PMMoV in concentrated samples (copies/reaction).

The recovery load of viruses (N [copies]) was calculated using Eq.(4):

where V concentrate represents the concentrate volume.

All statistical analyses were performed using R 3.6.0 (R Core Team, 2019). A post hoc pairwise Wilcoxon rank-sum test was performed for the multiple comparisons of the log W or log concentrations of PMMoV among different PEG precipitation procedures using "pairwise.wilcox.test" function in {stats} package. Comparisons with a P-value < 0.05 were considered significant. Note that the inequality sign (<) was placed only when a statistically significant difference was observed. Otherwise, an approximate symbol (≈) was placed. Spearman's rank correlation coefficient was determined to assess the relationship between the recovery load of each virus using "rcorr" function in {Hmisc} package.

J o u r n a l P r e -p r o o f Journal Pre-proof 3.

Comparison of whole process recovery efficiency among different PEG precipitation procedures Figure 2 shows the whole process recovery efficiency of MHV and phi6 and the observed concentrations of indigenous PMMoV with each PEG precipitation procedure.

All data required for the calculation are also provided in the supplemental spreadsheet.

The whole process recovery efficiency of MHV differed with the following order:

L.Long < LS.Long ≈ F.Long < L.0 < L.Short, ranging from −3.16 ± 0.80 log (geometric mean of 0.070%) to −1.59 ± 0.19 log (2.6%). Similarly, the observed concentration of indigenous PMMoV differed with the following order L.Long < F.Long ≈ LS.Long, < L.Short < L.0, ranging from 8.91 ± 0.27 log (7.9 × 10 8 ) to 9.74 ± 0.15 log (5.5 × 10 9 ) copies/L. Given that the indigenous PMMoV concentration in unconcentrated wastewater samples was determined to be 9.50 ± 0.30 log (3.2 × 10 9 ) copies/L, the estimated whole process recovery efficiency of PMMoV ranged from −0.88 ± 0.17 log (7.6%) to −0.05 ± 0.27 log (89%). Note that the whole process recovery efficiency of PMMoV may have been overestimated compared with MHV and phi6 because the initial concentration was directly determined from the unconcentrated wastewater, which may contain substances that may inhibit the molecular process. In fact, the MNV 

Comparison of SARS-CoV-2 detection among different PEG precipitation procedures Table 1 Also, PMMoV is a single-stranded RNA virus and thus quantifiable along with SARS-CoV-2 without requiring an additional step (e.g., heat denaturation of double-stranded RNA of phi6). However, the whole process recovery efficiency of PMMoV seems to be higher than that of MHV (see section 3.1). Therefore, the observed PMMoV concentration cannot be directly used for the back-calculation of the SARS-CoV-2 concentration but can be used as a normalization factor. Specifically, the SARS-CoV-2 concentration divided by observed indigenous PMMoV concentration can correct for the differences in whole process efficiency among samples and may help WBE implementers better capture the time-series trend of SARS-CoV-2. Several other studies have also proposed the advantages of co-investigation of fecal viral markers, including PMMoV (Graham et al., 2020; Wolfe et al., 2021) and crAssphage (Wilder et al., 2021) .

The use of phi6 as a WPC needs to be further discussed despite their morphological similarities. A recent study showed a significantly lower whole process recovery of phi6 (i.e., 3.9-log lower recovery than HCoV OC43 (Pecson et al., 2021) ). Another study showed 1-2-log higher whole process recovery by ultrafiltration compared with MHV (Fernandez-Cassi et al., 2021) . The quantification method for double-stranded RNA virus (e.g., phi6) typically includes heat denaturation step, and the efficiency of denaturation depends on the temperature, time, and the ionic strength of the medium (Gendron et al., 2010; Steger et al., 1980) . Although 95℃ is one of the most frequently used temperatures for denaturation, higher temperatures have been shown to provide a higher observed concentration of phi6 (Gendron et al., 2010) . This implies that the heat denaturation step at 95℃ might not work well especially in case of higher ionic strength 3.4. Implications for PEG precipitation as a primary concentration method for SARS-CoV-2 Table 2 shows the operational parameters of PEG precipitation along with the whole process recovery efficiency of spiked SARS-CoV-2 or enveloped surrogate viruses in previous studies. As a general trend, wastewater samples are centrifuged or filtered to remove large particles. Subsequently, the supernatant is mixed with PEG6000 or PEG8000 and NaCl and then incubated overnight. Some studies have performed this with pH adjustment, shorter incubation time, and/or without particle separation step (Ahmed et al., 2020a; Barril et al., 2021; Pecson et al., 2021; Pérez-Cataluña et al., 2021b; Philo et al., 2021; Sapula et al., 2021) . The reported whole process recovery considerably varied from 0.001% to 78%, depending on the surrogate viruses, PEG precipitation procedures, and the downstream molecular process ( Table 2) .

The results of this study (MHV, 0.070%-2.6%; phi6, 0.078%-0.51%) were at a lower range compared to previously reported values.

One of the main explanations is the difference in the operational parameters. The present study showed that a difference in the procedure (e.g., incubation time) leads to significantly (up to 1.57 log) different recovery efficiency (Figure 2 ). Other studies suggested that omitting the particle separation step may lead to stronger inhibition during RT-qPCR, resulting in lower whole process recovery efficiency (Sapula et al., 2021) .

Additionally, the difference in wastewater quality may also lead to inconsistency in the whole process recovery. For example, the whole process recovery efficiency of phi6 J o u r n a l P r e -p r o o f Journal Pre-proof by L.Long was 0.078% in this study, while that in a previous study (Torii et al., 2021) ranged 1.4%-3.0%. This is due to the lower molecular process recovery efficiency of MNV (−0.92 log) (Figure 3 ) in the present study compared with that in our previous study (−0.02 to 0.07 log) (Torii et al., 2021) .

Another potential explanation is an element of bias in the determination of the load of seeded viruses in each study (Kantor et al., 2021) , partially due to the selection of RNA extraction kit or method of determining the recovery efficiency. A previous study showed differences in the recovery of some enteric viruses with different RNA extraction kits (Ahmed et al., 2021; Iker et al., 2013) . This was also confirmed by our investigation (see Figure S1 ); the loads of seeded viruses differed depending on the RNA extraction kit. A review suggested that direct quantification from the viral stock leads to biased results due to the inhibition effect or presence of free RNA (Rusiñol et al., 2020) . Note that, in the present study, the positive control for the recovery test was prepared by spiking the viral stock into MilliQ water to achieve the same concentration in the wastewater sample.

Overall, the whole process recovery efficiency of PEG precipitation varied not only due to different operational parameters (such as incubation time and pretreatment) but also due to the differences in wastewater quality and bias resulting from the determination of the seeded load of viruses. Therefore, it is recommended to make the step-by-step procedure consistent to minimize quantification bias. The mean or range of whole process recovery efficiency in each study was reported.

Approx. was placed if the whole process recovery was not reported numerically but reported in the figure.

g Not NaCl but MgSO 4 was used as salt in the study.

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