MANUAL FOR DETECTIONOF
SARS-CoV-2 RNA IN WASTEWATER

COVID-19 Taskforce, Japan Society on Water Environment
Japan Institute of Wastewater Engineering and Technology
February 2022

EDITED BY
COVID-19 Taskforce, Japan Society on Water Environment
AUTHORS:
Tatsuo Omura (New Industry Creation Hatchery Center, Tohoku University)
Masaru Ihara (Graduate School of Engineering, Kyoto University)
Hiroyuki Katayama (Graduate School of Engineering, The University of Tokyo)
Masaaki Kitajima (Faculty of Engineering, Hokkaido University)
Daisuke Sano (Graduate School of Environmental Studies, Tohoku University)
Hiroaki Tanaka (Graduate School of Engineering, Kyoto University)
Shotaro Torii (Graduate School of Engineering, The University of Tokyo)
Akihiko Hata (Faculty of Engineering, Toyama Prefectural University)
Eiji Haramoto (Graduate Faculty of Interdisciplinary Research, University of Yamanashi)
Ryo Honda (Faculty of Geosciences and Civil Engineering, Kanazawa University)
Toru Watanabe (Faculty of Agriculture, Yamagata University)

The original version of this manual was published in Japanese by Japan Society on Water
Environment on 30 March 2021 (https://www.jswe.or.jp/aboutus/pdf/SARS-CoV2_RNA_Detection_Manual_for_Wastewater.pdf). This manual is its translated version in
English, and may contain old information which has been updated after publication of theoriginal version. The authors’ affiliations are at the time of publication of the original version.
The original manual and this translated version were edited as part of research projects
commissioned to the Japan Society on Water Environment by the Japan Institute of Wastewater
Engineering and Technology.
WebsiteJapan Institute of Wastewater Engineering and Technology
https://www.jiwet.or.jp/en
Japan Society on Water Environment
https://www.jswe.or.jp/eng/TABLE OF CONTENTS
1. INTRODUCTION

1

2. COLLECTION AND STORAGE OF SAMPLES
2.1 Sample Collection
2.2 Transport and Storage

222

3. ANALYTICAL METHODS
3.1 Virus Concentration
3.1.1 Process control
3.1.2 Concentration method
3.2 Detection of viral RNA by quantitative PCR
3.2.1 RNA extraction
3.2.2 Detection by RT-qPCR

4
4
4
10
15
15
17

4. SAFETY MANAGEMENT IN SAMPLE HANDLING
4.1 Handling of wastewater samples
4.2 Safety management in laboratory work for wastewater analysis

20
21
22

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

1. INTRODUCTION
The worldwide epidemic of novel coronavirus infections (COVID-19) has caused not only
loss of life and damage to health, but also tremendous damage to socioeconomic activities. The
main routes of transmission of the new coronavirus (SARS-CoV-2) are human-to-human droplet
and contact infections, but since viral genes can also be detected in human feces, detection of thevirus in sewage has been attempted worldwide. The measurement of the SARS-CoV-2 gene insewage may be able to reflect the prevalence of the disease in the treated area.
In this paper, we describe a method for the determination of SARS-CoV-2 in sewage. In
addition to the accuracy and sensitivity of the method, the cost of consumables, human resources,
equipment, speed, and efficiency should be taken into consideration. Therefore, the purpose ofthis paper is to give examples of various effective measurement methods so that they can bereferred to when selecting a measurement method. For this reason, we have listed the methods
that are currently recommended based on the results of previous studies.
Note that the last step in the detection of SARS-CoV-2 is assumed to be the PCR method,which detects viral genes and does not mean the presence of infectious viral particles. Therefore,
it should be noted that the results can be used as information on the prevalence of the disease in
the treated area, but they do not represent the risk of infection in sewage.

1

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

2. COLLECTION AND STORAGE OF SAMPLES
2.1 Sample Collection
The required volume of a wastewater sample depends on the applied concentration method(see details in Chapter 3). The typical volume of a sample is 100-250 mL for untreatedwastewater; 1.0-10 L for secondary treatment effluent or final effluent of a wastewater treatment
plant (WWTP). In collection of treated effluent after chlorine, residue chlorine should be
eliminated to prevent degradation of nucleotides in the sample. A concentrated sodium thiosulfate(Na2S2O3) solution should be added to the sample container in advance, then mixed well with thesample to the final concentration at approximate 50 mg/L.
A sealable container made of plastic or glass can be used as a sample container. The container
should be sterile or autoclaved before sample collection. If a sterilized container or autoclave is
not available, rinse the inside of the container with 1% sodium hypochlorite, leave it for a few
minutes, and then rinse very well with a sufficient amount of pure water at least five times toeliminate chlorine completely.
When collecting samples, wear the appropriate personal protective equipment (PPE).
Wearing masks and gloves is mandatory in most cases. If there is a possibility of splash exposure,consider necessity of face protection (safety goggles or face shields) and/or protective clothing.
After the sample collection, wash the surfaces of the sample container with tap water, thendisinfect it with ethanol or 5% chlorine bleach. Wash and disinfect hands immediately after
removing protective equipment. (see details in Chapter 4).
2.2 Transport and Storage
A sample should be stored and transported in a sealed container. If the sample is to beanalyzed within 24 hours, it should be transported on ice and stored in a refrigerator (below 10°C).
If the sample is to be analyzed later, it should be stored in an ultralow freezer at -80°C. If an
ultralow freezer is not available, store it in a laboratory freezer at -20°C or in a kitchen freezer,then analyze as soon as possible.
When a samples is transported from the sampling site to an external facility, it isrecommended to be transported in a triple-layer package (Figure 1) in a cool or freezing condition.
In the triple-layer packaging, the sealed primary container (= the sample container) should beplaced in an airtight secondary container (e.g. sealed plastic bag, Bio-pouch, Ziplock, etc.) withcushioning and leak absorbent material (e.g. paper towels, Kimtowel etc.). The secondary
container should then be placed in a tertiary container (e.g., foam box or cardboard box) that has
sufficient physical strength durable for the transport.
In order to ensure the safety during transportation and storage, a sample can be heatinactivated in a thermostatic bath at 56-60°C for 30-60 minutes1. The heat inactivation treatment
is effective to reduce the potential risk of infection; however, it does not guarantee complete
inactivation. Hence, it is highly recommended to take the same safety management regardless ofapplication of inactivation, for sample handling during transport, storage and sample analysis.
Decrease of SARS-CoV-2 RNA by heat inactivation under the above temperature and time
conditions is reportedly limited 1.

Pastorino, B., Touret, F., Gilles, M., de Lamballerie, X., Charrel, R.N., 2020. Heat Inactivation ofDifferent Types of SARS-CoV-2 Samples: What Protocols for Biosafety, Molecular Detection and
Serological Diagnostics? Viruses 12, 735. doi:10.3390/v12070735
1

2

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

Figure 1 Triple-layer packaging for transporting wastewater samples

3

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

3. ANALYTICAL METHODS
3.1 Virus Concentration
3.1.1 Process control
In the process of viral enrichment and detection, some effects cause inhibition, which
prevents There are several well-known Inhibitory effects on the virus recovery and detection fromsewage samples, which prevents accurate quantification of the viral gene. The inhibitory effectincludes the loss of viral particle/genome during the virus recovery and viral gene extraction,
decreased efficiency of the reverse transcription reaction and gene amplification in PCR. If
significant inhibition is detected, the obtained data are not available and virus recovery needs tobe repeated. Some surrogate viruses are available as process control viruses to detect the inhibition.
Of the four viruses listed below, all but pepper mild mottle virus (PMMoV) and F-phage areprepared in advance in the laboratory, and a known amount is added to the sample before the virus
recovery process (or the amount present is quantified), and the final percentage recovered is
calculated. In case of using PMMoV or F-phage, the recovery rate is calculated by measuring theconcentration before and after the recovery process, since the concentration of PMMoV or Fphage is originally high in sewage (note that inhibition after RNA extraction cannot be detected
when using F-phage). A recovery of 1% or higher may be considered acceptable (i.e., the recovery
process will not be repeated or the data will not be rejected). In general, the recovery rate of theprocess control virus is not used to convert the viral gene concentration of the target.
The following is a list of four viruses that can be used as process control viruses. Results
obtained without using at least one of these viruses or a virus equivalent to one of these viruses
as a process control virus may include a false negative result (a negative result due to inhibitioneven though the result is not really negative). If a process control virus is not used, the possibility
that the negative result is a false negative cannot be denied, and therefore the quantified value
should not be adopted as an official result. Even if the methods of recovery, RNA extraction,
reverse transcription, and real-time PCR are not completely consistent with this manual, there is
no problem in adopting the results obtained if the recovery rate of a process control virus isobtained without any problems (as for PMMoV, confirmation of the detected concentration fromsewage is acceptable).

4

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER(1) Murine hepatitis virus
Murine hepatitis virus (MHV) is a positive-sense single-stranded RNA virus belonging to
the genus Betacoronavirus, the same genus as SARS-CoV-2, which infects mice but is notpathogenic to humans and can be handled in the BSL2 facility. Several MHV strains are available
from ATCC (e.g., A59, VR-764), and MHV can be cultured in L2 cells or DBT cells2.
Several qPCR systems for MHV have been reported, and RNA can be quantified by RTqPCR. Here, we introduce the system of Besselsen et al. (2002)3, which is relatively widely used,
as an example. The sequences of primers and probe are as shown in Table 1.
Table 1 Sequences of primers and probe for qPCR of MHV4
Primers and probes
Sequence（5’–3’）
Primer (+)
GGAACTTCTCGTTGGGCATTATACT
Primer (-)
ACCACAAGATTATCATTTTCACAACATA
Probe
FAM-ACATGCTACGGCTCGTGTAACCGAACTGT-BHQ1
As a standard for calibration, use artificial synthetic DNA with the following sequence,
which is the target region of real-time PCR in MHV-A59 strain (ATCC accession number:
X00509).
GGAACTCTCGTGTGGGCATATATACTCTACTCTATATACATACATACATACATACAGCCGGTAGCATGTGTATATATATGTTGTTGTGAAAATGATAATCTTGTGGT
For the qPCR reaction conditions, as an example, Besselsen et al. (2002)3 used 40 cycles(95°C, 15 s → 60°C, 1 min).
(2) Bacteriophage φ6Bacteriophage φ6 is an enveloped double-stranded RNA virus, with a diameter of 85 nm.
Bacteriophage φ6 belongs to the family Cystoviridae and infects gram-negative Pseudomonas
syringae. This virus has been frequently used as a surrogate for pathogenic enveloped viruses (i.e.,
human coronavirus and influenza virus). A recent study adopted bacteriophage φ6 as a MPC.
Moreover, φ6 and its host, Pseudomonas syringae, are not pathogenic to humans and require
minimal biosafety level (i.e., BSL1). Bacteriphage φ6 can be propagated within one day with hightiters (up to 1010 PFU/mL); thus, φ6 is practically adoptable as a surrogate for the humanpathogenic enveloped viruses.
However, φ6 has double-stranded RNA and an envelope derived from Pseudomonas
syringae, which do not fully reflect the characteristics of SARS-CoV-2. The applicability of φ6
as a surrogate virus for process control needs further investigation.

2

Leibowitz et al. (2011) Coronaviruses: Propagation, quantification, storage, and construction of recombinant Mouse
Hepatitis Virus. Current Protocols in Microbiology, 15E.1.1-15E.1.46.
3
Besselsen et al. (2002) Detection of rodent coronaviruses by use of fluorogenic reverse transcriptase- polymerase
chain reaction analysis. Comparative Medicine, 52(2), 111-116.
4
Pastorino et al. (2020). Heat inactivation of different types of SARS-CoV-2 samples: What protocols for biosafety,
molecular detection and serological diagnostics? Viruses, 12(7), 735.

5

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

Table 2 Advantages and limitations of the application of the φ6 virus as a surrogate virus forprocess control
Advantages
Limitations
Morphological similarity to SARS-CoV-2
The presence of double-stranded RNA
Possess an envelope and thus exhibit
Additional steps (e.g., heat denaturation ofsimilar adsorption properties to mouse dsRNA before reverse transcription) arehepatitis virus (MHV) compared to MS2, a required in the process of quantification.
non-enveloped virus.
No explicit proof of comparability to
Can be used in BSL1-compliant facilities.
SARS-CoV-2.
Not required for BSL2 facilities (BSL2 is
The structural differences between φ6 andrequired for handling envelope surrogate SARS-CoV-2 may affect the recovery
viruses such as MHV).
efficiency.
Easy to culture
Pseudomonas syringae and φ6 can beeasily cultured at high concentrations.
a) Reagents and instruments
Culture・ Pseudomonas syringae: Available at the National Institute of Technology and Evaluation(NITE). (NBRC 14084)
・ Bacteriophage φ6: Available at NITE. (NBRC 105889)
PCR
Primer and probe: The sequence is shown in Table 3.
Table 3 Sequences of primers and probes for qPCR of φ6 (Gendron et al. 2010)
Primers and probes
Sequence （5’–3’）
Primer (+)
TGGCGGCGGTCAAGAGC
Primers (-)
GGATGATTCTCCAGAAGCTGCTG
Probe
FAM-CGGTCGTCGCAGGTCTGACACTCGC-BHQ1
Dilute the standard with TE buffer, etc. as appropriate.
TGGCGGCGGTCAAGAGCAACCCGGTCGTCGCAGGTCTGACACTCGCTCAG
ATCGGAAGCACCGGTTATGACGCCTATCAGCAGCTTCTGGAGAATCATCC(3) F-phage
F-specific bacteriophages (F-phages) are viruses that infect E.coli possessing F-pili. Fphages multiply in humans and other animals, and are excreted with feces. F-phages are classified
into FDNA and FRNA phages. The former possess single stranded DNA in a rod-shaped capsid,
while the latter possess single-stranded RNA in an icosahedral capsid with a diameter of 20-30
nm, like typical enteric viruses. FRNA phages are classified into four genogroups/serogroups of
GI-GIV, which are represented by MS2, GA, Qβ, and SP phages, respectively. Both FDNA and
FRNA phages are non-enveloped viruses.
F-phages in sewage can be detected and quantified by a cultural method. Advantages of using
F-phage as a process control are that it does not require the addition of virus to the sample, andthat with the cultural method, its quantification can be conducted at low cost. In addition, since
nucleic acid extraction and RT-qPCR are not required, loss of RNA or RT-qPCR inhibition that
potentially occur during these processes do not matter, and only the recovery efficiency during
the concentration process can be estimated. On the other hand, disadvantages of using F-phage
are that it takes overnight to obtain results and that the recovery efficiency of F-phage does notnecessarily reflect that of SARS-CoV-2 because F-phage is a non-enveloped virus. Considering
the diversity of F-phage, the recovery efficiency may vary depending on the genotype or strain.
6

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

In addition, since the detection is based on the cultural method, F-phage is not applicable when
the sample is inactivated. Its concentration may decrease significantly when the sample is stored
for a long time.
In the following, we introduce a plate counting method (plaque assay) using Salmonella
typhimurium WG49 as the host, for the quantitative detection of F-phage. BSL2 laboratory is
required for the use of Salmonella typhimurium WG49.
a) Reagents and instruments
・ Petri dish: Use a sterilized one.
・ Salmonella typhimurium WG49 (ATCC® 700730™): Salmonella typhimurium WG49
should be handled in the BSL2 laboratory. Prepare a glycerol stock and store at -80°C (see"Preparation of a glycerol stock for Salmonella typhimurium WG49" below). Medium andpetri dish containing Salmonella typhimurium WG49 should be discarded after being
autoclaved.
・ Glycerol: Use for making glycerol stock.
・ Trypton: Thermo Fisher Scientific, 211705, etc. Use as a component of culture medium.
・ D(+)-Glucose: FUJIFILM Wako Pure Chemical, 047-31161, etc. Use as a component of
culture medium.
・ CaCl2 ・2H2O: Use as a component of the culture medium. Prepare 0.3 g/mL calcium
chloride solution by mixing 0.40 g of the reagent with 1 mL of purified or ultrapure water.
・ MgSO4・7H2O: Use as a component of the culture medium. Prepare 0.15 g/mL magnesium
sulfate solution by mixing 0.31 g of the reagent with 1 mL of purified or ultrapure water.
・ Agar: Bacto™ Agar (BD, 214010), etc. Use as a component of the culture medium.
・ Kanamycin sulfate: Sigma Aldrich, K4000-5G, etc. Use as a component of the culture
medium. Prepare an aqueous solution of 20 g/L and keep frozen in advance.
・ Nalidixic acid sodium salt: Sigma Aldrich, N4382-5G, etc. Use as a component of the culture
medium. Prepare an aqueous solution of 100 g/L and keep frozen in advance.
・ Liquid medium: Prepare by mixing the reagents according to Table 4 and autoclave. Use forculture of Salmonella typhimurium WG49.
・ Agar medium: Prepare by mixing reagents according to Table 4 and autoclave. Can be stored
at 50°C without solidification.
・ Phosphate buffer: FUJIFILM Wako Pure Chemical, 161-12191, etc. Use for dilution ofsamples.
・ Incubator: Set at about 37°C (as in the case of detection of E. coli, etc.).
Table 4 Composition of liquid medium and agar medium used for F-phage detection
Reagents
Unit Liquid medium Agar medium
Purified water or ultrapure water
mL
500
500
Tryptong
5
5
D(+)-Glucose
g
0.5
0.5
NaCl
g
4
4
CaCl2 (aq) (adjusted to 0.3 g/mL)
mL
0.5
0.5
MgSO4 (aq) (adjusted to 0.15 g/mL)
mL
0.5
0.5
Bacto Agarg
5.5
Kanamycin sulfate (aq) (adjusted to 20 g/L)
mL
0.5※
Nalidixic acid sodium salt (aq) (adjusted to 100 g/L)
mL
0.5※
Salmonella typhimurium WG49 after liquid culturemL
20※※
Kanamycin, nalidixic acid, and Salmonella typhimurium WG49 should be added immediately
before plating.
7

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATERb) Procedure
Preparation of a glycerol stock for Salmonella typhimurium WG49.
1. Dilute 63 g of 100% glycerol with pure water to be 100 mL of 50% glycerol.
2. Autoclave the 50% glycerol and bring to room temperature.
3. Add a glycerol stock of Salmonella typhimurium WG49 to the 100× volume of liquidmedium, and incubate in an incubator at 37°C for about 4 hours with shaking.
4. Check the turbidity of the liquid medium visually after the incubation. If there is no
turbidity, continue the incubation for another 1-2 hours.
5. Mix 100 μL of the liquid medium after completing the incubation with 100 μL of 50%
glycerol in a cryo tube and store in a deep freezer (-80°C).
Detection of F-phage from wastewater samples
1. Prepare agar medium, assuming about 20 mL per Petri dish. Keep the medium at 50°C until
just before use to prevent it from solidifying.
2. Prepare 20 mL of the liquid medium per 500 mL of agar medium.
3. Thaw the glycerol stock of Salmonella typhimurium WG49.
4. Add a glycerol stock of Salmonella typhimurium WG49 to the 100× volume of liquidmedium, and incubate in an incubator at 37°C for about 4 hours with shaking. Use amedium in the logarithmic growth phase. The turbidity of the liquid medium can help todetermine if it is in the logarithmic growth phase or not.
5. Spread the sample in a Petri dish. Spreading volumes of 100 μL or 1 mL per Petri dish areappropriate. Prepare multiple sample volumes in 10-fold steps in duplicate so that at least
one of them results in 10 to 100 plaques (number of F-phages) per Petri dish. If < 100 μL
sample volume needs be tested, dilute the sample with phosphate buffer in 10-fold steps.
In the case of concentrated solution, the sample volume can be determined based on thevolume reduction rate by concentration process and the expected recovery rate (1 to 100%).
6. Add Salmonella typhimurium WG49, kanamycin sulfate (aq), and nalidixic acid sodium
salt (aq) to agar medium (preferably at 45°C, which is safe to grip with hands).
7. Add an appropriate amount of agar medium to each Petri dish and allow it to solidify atroom temperature.
8. Incubate the Petri dish in an incubator at 37°C overnight.
9. Select a Petri dish with a sample volume where 10 to 100 plaques formed and count theplaques.
10. Calculate the concentration of F-phage in the sample based on the plaque count. For
example, if 40 and 60 plaques in a sample volume of 100 μL (in duplicate) are obtained,
the concentration of F-phage is determined as 500 PFU/mL.
11. Dispose the Petri dish and agar medium after autoclaving.
(4) Pepper mild mottle virus
Pepper mild mottle virus (PMMoV), a plant virus that infects peppers and other peppers of
the genus Capsicum, is known to be present at extremely high concentrations in human feces andhas been detected at high frequencies and concentrations in wastewater and environmental water
worldwide 5 . Since the concentrations of PMMoV in wastewater don’t show clear seasonalvariations and are relatively constant, the measurement of indigenous PMMoV in wastewater
samples can be used to confirm that no serious problems are occurring during the detection
procedures. The advantage of using this process control is that it does not require adding PMMoV
to a sample.
5

Kitajima et al. (2018) Pepper mild mottle virus as a water quality indicator. npj Clean Water. 1:19.

8

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATERa) Reagents・ Forward primer, reverse primer, TaqMan MGB probe: Use oligo DNA with the sequences
shown in Table 5. Note that the quencher dye of the probe is NFQ-MGB, not TAMRA or
BHQ.
・ Standard sample: Use artificially synthesized DNA, etc., with the following sequencecorresponding to the qPCR amplification region for PMMoV (ATCC accession number:
NC_003630). Dilute with TE buffer or other appropriate solution.
GAGTGGTGTGACCTTAACGTTGTGAGGCCTACGACGAAGCAATGTCGCACTTGCATTGCAACCGACAA
Table 5 Sequences of primers and probe for qPCR of PMMoV6,7
Primer/probe
Sequence (5’–3’)
Forward primer
GAGTGGTTTGACCTTAACGTTTGA
Reverse primer
TTGTCGGTTGCAATGCAAGT
TaqMan MGB probe
FAM-CCTACCGAAGCAAATG-NFQ-MGBb) Procedure
1. Obtain viral RNA extract by virus concentration and RNA extraction steps, without adding
PMMoV to the sample.
2. In two-step RT-qPCR, obtain cDNA sample by RT, following the components of RT
mixture and thermal conditions described in the protocol of the reagent.
3. In two-step RT-qPCR, prepare qPCR mixture, while in one-step RT-PCR, prepare RTqPCR mixture. Mix the mixture with cDNA or RNA extract for the detection of PMMoV.
Use the primers and probe listed in Table 5. Follow the components of the mixture andthermal conditions shown in the protocol of the reagent; however, the conditions may need
to be optimized in case that amplification efficiency is not high.
4. In both one- and two-step RT-qPCR, instead of cDNA or RNA extract, use a standard
sample diluted with TE buffer, etc., to be detected with a Ct value of approx. 25–30 as apositive control and PCR-grade water as a negative control. For quantification, prepare 10fold serial dilutions of the standard sample diluted with TE buffer, etc., and draw a standard
curve within the range of 101 to 106 copies/reaction.
5. Determine a Ct value of the tested sample using the software. If there is no significant
difference from previous qPCR results, it is judged that there was no serious problem in thedetection procedure.
6. Recovery of PMMoV can be calculated by storing approx. 1 mL of the wastewater sample
before the concentration step, quantifying PMMoV in both the sample and the concentrated
sample, and then calculating a ratio of the amount of PMMoV in the original wastewater
sample to that in the concentrated sample by considering several factors, such as thevolumes of the samples.

6

Haramoto et al. (2013) Occurrence of pepper mild mottle virus in drinking water sources in Japan. App. Environ.
Microbiol. 79 (23), 7413–7418.
7
Zhang et al. (2006) RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biol. 4 (1),0108–0118.

9

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

3.1.2 Concentration method(1) Polyethylene glycol precipitation method
Polyethylene glycol (PEG) precipitation method is widely used for the concentration ofprotein components, including viruses. The method starts from mixing polyethylene glycol (awater-soluble polymer) and sodium chloride with wastewater sample. Incubation of the mixture
allows the polyethylene glycol for associating with the proteins in the sample. Aftercentrifuging the mixture, the supernatant is discarded. The remaining pellets are resuspended ina small amount of buffer solution and subjected to downstream processing. This method has
been used as a primary concentration method for untreated sewage samples and as a secondary
concentration method for other water samples. PEG precipitation method can reduce the volume
of liquid from 100 mL to about 1 mL or less. PEG with a molecular weight of 6,000 or 8,000(PEG6000 and PEG8000, respectively) along with sodium chloride is typically added to thewater sample. Several studies optimized the type of PEG, the concentration of PEG and sodium
chloride, mixing conditions (temperature and time), and centrifugation conditions for improving
the efficiency of virus concentration. Here, the method proposed by Jones and Johns (2009) is
introduced as an example.
a) Equipment
・A shaker with a cooling function or a shaker that can be installed in a refrigerator, etc.
・Centrifuge with cooling function: A centrifuge capable of centrifuging 50-mL tubes at 10,000
x g. A fixed angle rotor is preferable.
b) Reagents and instruments
・PEG8000: Wako Pure Chemicals, 593-09765, etc.
・Sodium chloride: Wako Pure Chemical Industries, 191-01665, etc.
・Sterilized 50-mL centrifuge tubes
・Sterilized 50-mL centrifuge tube for PEG precipitation: Add 4.0 g of PEG8000 and 2.35 g of
NaCl in advance.
・Phosphate buffer (PB): Wako Pure Chemicals, 161-12191, etc.
・1.5 mL tube: Used for collecting the concentrated solution.
c) Procedure
The method is shown for concentrating 80 ml of sample using two centrifuge tubes, each ofwhich contains 40 mL of samples.
1. Add 4 g of PEG8000 and 2.35 g of NaCl to the centrifuge tube. Close the lid.
2. Add 40 mL of the sample into two empty 50-mL tubes.
3. (In the case of adding a surrogate virus for process control) After adding the surrogate virus,
incubate the samples in a refrigerator for at least 1 hour until a solid-liquid equilibrium isreached between the surrogate virus and the sewage components.
4. Centrifuge at 3,500 x g for 5 min and transfer the supernatant to a centrifuge tube containing
PEG and NaCl. Shake the tube gently so that PEG and NaCl do not remain undissolved at
the bottom. Of note, since a large proportion of SARS-CoV-2 RNA in sewage may exist in
solid-phase, the centrifugation (solid-liquid separation) may be omitted. It is also possible toextract RNA from the pellet and analyze it separately from the supernatant.
5. Incubate the mixture overnight at 4 °C in a shaker.
6. Centrifuge the mixture of the sample, PEG, and NaCl at 10,000 × g for 30 min.
7. Discard the supernatant. Resuspend the precipitates in 500 µL of phosphate buffer. Since
the precipitate adheres to the wall of the tube, try to resuspend as much as possible bypipetting. Instead of phosphate buffer, purified water, ultrapure water or TRIzol Reagent(Invitrogen, 15596018, etc.) can be used.
10

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

8. Centrifuge the tube for a few seconds to collect the suspension at the bottom.
9. Transfer all the suspension to a 1.5 mL tube. Measure the final volume. The final volume isusually around 0.5 to 1 mL.
10. Subject the concentrates to RNA extraction. If no further RNA extraction is to be
performed, store the virus concentrate at -20°C or lower.
Note that several other PEG precipitation conditions have been reported. For example, Wu
et al. (2020) successfully detected SARS-CoV-2 RNA by using 0.22 μm PES membrane
filtration, salt concentration of 0.3 M, shaking time of 15 minutes, and centrifugation time of 2hours in the solid-liquid separation process.
(2) Electronegative membrane-vortex method
Electronegative membrane-vortex (EMV) method uses an electronegative membrane filter,
which has been widely used for the concentration of enteric viruses, to adsorb viruses to themembrane by electrostatic attraction in the presence of cations, and to recover viruses alongwith turbidity by vigorous vortex mixing of the membrane in an elution buffer8. A centrifugal
filter unit can be used as a secondary concentration method. The EMV method has been used
for the concentration of enteric viruses, such as noroviruses and PMMoV, one of the processcontrol viruses, in various types of water samples, such as wastewater, river water, andgroundwater. This method has also been used for the successful detection of SARS-CoV-2
RNA at a wastewater treatment plant in Yamanashi Prefecture, Japan9. In addition, this method
can simultaneously concentrate not only viruses but also protozoa (Cryptosporidium, Giardia,etc.) and bacteria by using pellet portions after centrifugation.
a) Equipment
・ Suction filtration pump.
・ Filtering flask.
・ Glass filter holder (diameter, 90 mm): Cover the open parts with aluminum foil when
autoclaving. Advantec, cat. no. KG-90, or equivalent.
・ Centrifuge: A centrifuge capable of cooling to 4°C is desirable, but a cooling function is notessential.
・ Swing rotor: A rotor that can centrifuge 50-mL centrifuge tubes at a maximum of approx.
2,000 × g. When using a fixed-angle rotor, it is desirable to consider the optimum conditions
for centrifugation prior to the analysis. A fixed-angle rotor is also required if Amicon Ultra15 is used as a secondary concentration method.
b) Reagents and consumables
・ Mixed cellulose membrane (diameter, 90 mm; pore size, 0.8 µm): Mixed cellulose
membrane consisting of cellulose ester and nitrocellulose (Merck Millipore, cat. no.
AAWP09000, or equivalent). The membrane is placed in a glass filter holder, and the open
portions are covered with aluminum foil. After autoclaving, the glass filter holder is storedwith the aluminum foil attached.
・ MgCl2 solution (2.5 mol/L): Prepare by dissolving MgCl2 6H2O in (ultra)pure water,
autoclave, and store at room temperature. Add 1 mL of this solution per 100 mL of water
sample.
8

Haramoto et al. (2012) Development of a novel method for simultaneous concentration of viruses and protozoa froma single water sample. J. Virol. Methods 182, 62–69.
9
Haramoto et al. (2020) First environmental surveillance for the presence of SARS-CoV-2 RNA in wastewater andriver water in Japan. Sci. Total Environ. 731, 140405.

11

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

・ Football-shaped stirring bar (25 × φ10 mm): As One, cat. no. 7-217-01, or equivalent.
・ Elution buffer (100 ×): 2 g of Na4P2O7 10H2O (Kanto Chemical, cat. no. 37256-00, or
equivalent), 3g of C10H13N2O8Na3 3H2O (Wako Pure Chemical, cat. no. 342-01875, or
equivalent), and 1 mL of Tween 80 (Sigma-Aldrich, cat. no. P1754-25ML, or equivalent) inapprox. 70 mL of (ultra)pure water, adjust pH to 7.2 by adding HCl, scale-up to 100 mL,
autoclave, and store in a refrigerator.
・ Elution buffer (1 ×): Prepare 100 times dilution of the elution buffer (100 ×) using(ultra)pure water, autoclave, and store in a refrigerator.
・ Tweezer: Wrap in aluminum foil and autoclave.
・ Transfer pipette: Sterilized and disposable.
・ Centrifugal filter units: Centriprep YM-50 (Merck Millipore, cat. no. 4311 (96 units)),
Amicon Ultra-15 (Merck Millipore, cat. no. UFC905008 (8 units)), or equivalent.
・ Disposable filter unit (pore size, 0.45 μm; diameter, 25 mm): Advantec, cat. no.
25CS045AS, or equivalent.
・ Syringe (volume, approx. 20 mL): Terumo, cat. no. SS-20ESZ, or equivalent.
・ Electronic pipette: To be used by attaching a plastic pipette. Nichiryo, cat. no. NEO00PMNEO, or equivalent.
・ Plastic pipette: Sterilized. Preparing multiple sizes, such as 10 or 25 mL, is recommended.
・ Micropipette: Sterilized.
・ Micropipette tip: Sterilized. 20, 200, or 1000 μL, etc. Filter tips are preferable.
・ Microtube: Sterilized. 1.5 or 2.0 mL, etc.
・ Plastic paraffin film: Parafilm (As One, cat. no. 6-711-01), etc.
c) Procedure
1. Add 1 mL of MgCl2 solution (2.5 mol/L) per 100 mL of water sample, mix by inverting,and incubate it at room temperature for a few minutes.
2. Filter the water sample through a glass filter holder attached with a 90-mm diameter mixed
cellulose membrane. Normally, filtration of 200 mL is possible for influent wastewater.
However, since filtration may be difficult depending on water quality, adjust the filtration
volume while checking for clogging. When testing treated or effluent wastewater, filtrationof approx. 5 to 10 liters is possible. When using membranes with a diameter of 47 mm, thestandard filtration volume is approx. 50 mL for inflow sewage and 1 to 2 L for treated andeffluent wastewater. Meanwhile, when using 47-mm membranes, two or more membranes
may be used to filter a single water sample, but no more than four membranes may be used.
3. Remove the filtered membrane from the filter holder with a tweezer, fold it, and transfer it
to a 50-mL centrifuge tube containing 10 mL of eluent buffer and a football-shaped stirringbar. When more than two membranes are used for filtration, all membranes should beplaced in a single centrifuge tube. When Amicon Ultra-15 is used as a secondary
concentration method, the volume of the elution buffer should be 8 mL because there is apossibility of sample leakage during centrifugation. Similarly, when using one 47-mm
membrane, the volume of the elution buffer can be set to 8 mL to reduce the volume of the
final concentrated solution.
4. Wrap a plastic paraffin film around the cap of the centrifuge tube.
5. Vortex the centrifuge tube vigorously with a vortex mixer to crush the membrane to a few
mm square or less. In order to avoid unevenness in mixing, invert and mix as necessary toensure uniform mixing. The membrane is sufficiently crushed in about 5 min.
6. Recover the whole volume of the solution using a transfer pipette and transfer it to a new
50-mL centrifuge tube. Be careful not to recover the crushed membrane as much as
possible.

12

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

7. Add 5 mL of the elution buffer (4 mL if 8 mL was used in the first elution step) to theoriginal tube, agitate with a vortex mixer for ~30 sec (plastic paraffin film is not necessary),
and transfer the whole volume of the solution with a transfer pipette to the above mentioned
tube. At this step, recover as much of the solution as possible from the inner wall of the tube
and from the inside of the cap. The final volume of the solution in the new tube will be ~15
mL (~12 mL if 8 mL was used in the first elution step).
8. Centrifuge the centrifuge tube using a swing rotor (2,000 × g, 10 min, 4°C), and recover thesupernatant with a transfer pipette. The temperature should be set at 4°C, but it is notnecessary. The brake of the centrifuge should be turned off.
9. Transfer the collected supernatant to a syringe equipped with a disposable filter unit andfilter under pressure to collect the filtrate. For collection of the filtrate, use the outer unit
when using Centriprep YM-50 or the inner unit when using Amicon Ultra-15.
10. The following is the procedure when Centriprep YM-50 or Amicon Ultra-15 is used as thesecondary concentration method. When using Centriprep YM-50, perform Steps 11–14, andwhen using Amicon Ultra-15, perform Steps 15–16. If a suitable centrifugal filter unit is notavailable, it is possible to skip the secondary concentration and use the concentrated sample
obtained in Step 9, although the detection sensitivity will be decreased due to insufficient
reduction of volume of the concentrate.
11. [Centriprep YM-50] Attach the inner unit of Centriprep YM-50 to the outer unit, andcentrifuge it using a swing rotor (2,000 × g, 10 min, 4°C).
12. [Centriprep YM-50] Confirm that the inner and outer units are firmly attached, remove thecap of the inner unit, turn it upside down, and discard the solution in the inner unit.
13. [Centriprep YM-50] Centrifuge Centriprep YM-50 again (2,000 × g, 5 min, 4°C) to further
reduce the sample volume in the outer unit.
14. [Centriprep YM-50] Remove the inner unit (the solution in the inner unit is not necessary),and recover the solution in the outer unit into a microtube (1.5 or 2.0 mL) using a
micropipette, by recording the volume of the sample. Record the sample volume in units of
10 μL. If the sample volume exceeds 1 mL, store only 1 mL as a concentrated sample anddiscard the remaining sample.
15. [Amicon Ultra-15] Centrifuge Amicon Ultra-15 (5,000 × g, 15 min, 4°C) with the inner unit
attached to the outer unit.
16. [Amicon Ultra-15] Recover the remaining sample in the inner unit into a microtube (1.5 or
2.0 mL) using a micropipette, by recording the sample volume. Record the sample volume
in units of 10 μL. If the sample volume exceeds 1 mL, store only 1 mL as a concentrated
sample and discard the remaining sample. Note that the total volume of the concentrated
sample should be recorded because it is necessary for calculating the concentration.
If no further RNA extraction is performed, store the virus concentrated sample at -20°C or
lower.
(3) Ultrafiltration membrane method
The ultrafiltration membrane method concentrates virus particles by a "sieving effect" using
an ultrafiltration membrane with a molecular weight cut-off of 10 to 100 kDa. The ultrafiltration
method has been used for the detection of SARS-CoV-2 in wastewater in the Netherlands10 and
the U.S.11, and has been successful in detecting the virus. The advantage of this method is that itis relatively simple to operate because it uses commercialized ultrafiltration membrane units (e.g.,
10Medema et al. (2020) Presence of SARS-Coronavirus-2 RNA in sewage and correlation with reported COVID-19
prevalence in the early stage of the epidemic in The Netherlands. Environ. Sci. Technol. Lett. 7, 511–516.
11
Sherchan et al. (2020) First detection of SARS-CoV-2 RNA in wastewater in North America: A study inLouisiana, USA. Sci. Total Environ. 743, 140621.

13

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

Centricon Plus-70 from Merck Millipore). On the other hand, the cost of disposable ultrafiltration
units is high (about 4,000 JPY/unit for Centricon Plus-70), and they tend to be suffered byinhibition in the subsequent molecular detection process because they simultaneously concentrateinhibitors in the wastewater. Here, we introduce a method using the Centricon Plus-70 from
Merck Millipore.
a) Required equipment
・ Filter holder for decompression filtration
・ Aspirator
・ Centrifuge with swinging rotor
・ Tweezersb) Reagents and instruments
・ Centricon Plus-70 100 kDa (Merck Millipore, model number: UFC710008)
・ Hydrophilic PTFE membrane (pore size: 0.2 µm)c) Procedure
1. Filter the wastewater sample through a hydrophilic PTFE membrane (pore size: 0.2 µm).
(However, since a significant proportion of SARS-CoV-2 in wastewater may be associatedwith suspended solids, the filtration (solid-liquid separation) step may be omitted unless thesuspended solids interfere with the subsequent molecular detection process.)
2. Using an ultrafiltration membrane (Centricon Plus-70), centrifuge 120 mL of the PTFE
membrane at 1,900 x g for 8 minutes twice, and then centrifuge it upside down at 800 x g for
2 minutes to collect the concentrate. This method enables concentration of 120 mL ofwastewater sample to about 400-700 µL.
3. If RNA extraction is not performed immediately, store the concentrate at -20°C or lower.

14

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

3.2 Detection of viral RNA by quantitative PCR
In order to confirm that there is no contamination at a level that affects the detection resultsin the operations after RNA extraction, when RNA extraction is performed, add water formolecular biology (RNase free water, e.g., Water for molecular biology (Merck, model number:
95284-100ML)) in the same volume as the wastewater concentrate as a negative control, andperform RT-qPCR for SARS-CoV-2 and a surrogate virus for process control. If either or both of
SARS-CoV-2 and the surrogate virus for process control are detected, contamination will haveoccurred in the operation after RNA extraction, and the results of SARS-CoV-2 RNA detection
will not be obtained. The detection result of SARS-CoV-2 RNA should not be used. Even when
this negative control is used, it is necessary to include a negative control for qPCR in order todetermine whether or not there is contamination in the qPCR process.
3.2.1 RNA extraction(1) Method using the nucleic acid extraction kit QIAamp Viral RNA Mini Kita) Necessary equipment
・ Vortex mixer
・ Microtube centrifuge (usable at 14,000 x g)b) Reagents and instruments
・ QIAamp Viral RNA Mini Kit (Qiagen, model number: 52906)
・ TRIzol Reagent (Thermo Fisher Scientific, model number:15596026)
・ Ethanol (96-100%)c) Procedure
Dispense 140 µL wastewater concentrate and 560 µL Buffer AVL into a 1.5 mL tube, pulse
vortex, and incubate at room temperature for 10 min. When the precipitate obtained by PEG
precipitation is resuspended in TRIzol Reagent, mix 140 μL of the concentrated wastewater
solution after pulse vortexing with 560 μL Buffer AVL, pulse vortex again, and incubate at room
temperature for 10 minutes. Add 560 µL ethanol to the sample and pulse vortex. Centrifuge at
6,000 x g (8,000 rpm) for 1 minute and discard the collection tube containing the filtrate. Afterall the samples have been applied to the column, add 500 µL of washing buffer (Buffer AW1) tothe column, centrifuge at 6,000 x g (8,000 rpm) for 1 minute, and discard the collection tube
containing the filtrate. Add another 500 µL of wash buffer (Buffer AW2), centrifuge at 20,000 x
g (14,000 rpm) for 3 minutes, and discard the collection tube containing the filtrate. Attach a newcollection tube to avoid carryover, and centrifuge at maximum speed for 1 minute. Place thecolumn in a new 1.5 mL tube, add 60 µL Buffer AVE, and centrifuge at 6,000 × g (8,000 rpm)
for 1 minute. The filtrate obtained by this operation is the RNA extraction solution. If the RT
reaction is not performed subsequently, store the RNA extract at a temperature below -20°C (or 80°C if possible).
(2) AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method
The AGPC method is an RNA extraction and purification method developed for isolating
total RNA from cells and tissues. TRIzol (Thermo Fisher Scientific) and TRI Reagent (Sigma
Aldrich), which contain phenol and guanidine isothiocyanate, are widely used to perform the
AGPC method. A slightly modified method, using TRIzol and a commercially available RNA
extraction kit, has also been proposed. Here, we introduce the TRIzol + RNeasy Mini Kit
method using TRIzol and the RNeasy Mini Kit.
Note that TRIZOL® LS contains phenol (toxic, corrosive) and guanidine isothiacyanate(irritant). The extraction should be performed with appropriate equipments.
1. [Addition of murine norovirus (MNV) as an option] Add 10 μL of MNV to 250 μL of PEG
concentrate. As a control sample, prepare 250 µL of MilliQ water spiked with 10 µL of
MNV.
15DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER2.

Add 750 µL of TRIzol® LS Reagent and homogenize the mixture for about 30 seconds
using Fast-prep 24. Vigorous vortex is also acceptable. Incubate at room temperature for 5
minutes.
3. Add 200 µL of chloroform and homogenize again for about 15 seconds. Incubate at room
temperature for 2-15 minutes.
4. Centrifuge at 12,000 x g for 15 minutes at 4°C.
The solution after centrifugation is separated into a dark red phenol-chloroform phase, anintermediate phase, and a colorless aqueous phase. The volume of the resulting aqueous
phase will be approximately ~70% of the TRIZOL® LS reagent used.
5. While waiting for centrifugation, prepare a 1.5 mL tube and dispense 650 µL of 70%
ethanol.
6. [Ethanol precipitation of RNA] Tilt the tube at 45 degrees and carefully collect the aqueous
phase, and transfer it to the prepared tube containing 70% ethanol. Mix well by pipetting.
7. [Adsorption of RNA to silica membrane] Put 700 μL of the mixture into a spin column.
Close the lid, and centrifuge at 8,000 × g for 15 seconds.
8. Discard the liquid in the collection tube, and add the remaining mixture (600-700 μL) to
the spin column again. Close the lid, and centrifuge at 8,000 × g for 15 seconds.
9. [Washing] Discard the liquid in the collection tube, and add 700 μL of Buffer RW1 to thespin column. Close the lid and centrifuge at 8,000 x g for 15 seconds.
10. [Washing] Discard the liquid in the collection tube and add 500 μL of Buffer RPE to thespin column. Close the lid and centrifuge at 8,000 x g for 15 seconds.
11. [Washing] Discard the liquid in the collection tube, and add 500 μL of Buffer RPE to thespin column. Close the lid and centrifuge at 8,000 x g for 2 minutes.
12. [Drying] Discard the liquid in the collection tube and centrifuge at 12,000 × g for 1 minute.
13. [RNA extraction] Place the spin column to a newly prepared 1.5 mL tube and add 30 µL of
RNase-free water to the silica membrane, incubate at room temperature for 1 min, andcentrifuge at 8,000 × g for 1 min.
14. [RNA extraction] Open the lid of the spin column again, add 30 μL of RNase-free water,
and perform incubation as in step 13. Then, centrifuge the column at 8,000 x g for 1 minuteto obtain a total of 60 μL of RNA extract. If the RT reaction is not performed subsequently,
store the RNA extract below -20°C (or -80°C if possible).
.

16

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

3.2.2 Detection by RT-qPCRa) Necessary equipment
・ Real-time PCR system (e.g., ABI 7500, Light Cycler 480)
・ PCR system (thermal cycler)b) Reagents and instruments
・ Reverse transcription kit (e.g., High Capacity cDNA Reverse Transcription Kit (Thermo
Fisher Scientific))・ Real-time PCR master mix (e.g. QuantiTect Probe qPCR kit (QIAGEN))・ 96 well real-time PCR reaction plate・ 8-strip cap or plate seal・ Primers (Table 6)
・ TaqMan probe (Table 6)・ RNase free water
Table 6 Sequences of primers and probes of qPCR assays for SARS-CoV-212
Assay
Primers / Probe
Name
Sequence
CDC N1 Primer (+)
2019-nCoV_N1-F GACCCCAAAATCAGCGAAAT
Primer (-)
2019-nCoV_N1-R TCTGGTTACTGCCAGTTGAATCTG
Probe
2019-nCoV_N1-P FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1
CDC N2 Primer (+)
2019-nCoV_N2-F TTACAAACATTGGCCGCAAA
Primer (-)
2019-nCoV_N2-R GCGCGACATTCCGAAGAA
Probe
2019-nCoV_N2-P FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1
In past domestic and international studies, the qPCR assays of CDC N1 and CDC N2 havebeen reported to be the most widely used systems for the detection of SARS-CoV-2 RNA fromwastewater samples and to show high detection sensitivity, so we recommend the use of theseqPCR assays. However, the use of other qPCR assays may be acceptable if a qPCR assay withsuperior detection sensitivity is announced with the progress of future research. In that case, theprimers and probes will be changed, but the basic detection procedure will be the same as themethod described in this manual.
c) Procedure
Reverse transcribed viral RNA into cDNA (complementary DNA) using reverse
transcriptase, and then quantify the cDNA by real-time PCR. As examples, the reaction conditions
for reverse transcription using Thermo Fisher Scientific’s High Capacity cDNA Reverse
Transcription Kit and real-time PCR using QIAGEN’s QuantiTect® Probe PCR kit are shown
below. The reaction conditions are shown below. For details, refer to the manual attached to thekit. All dispensing operations of reagents should be performed on ice.
Reverse transcription reaction
The RNA extract is mixed with the same volume of reverse transcription reaction solution
and used for the reverse transcription reaction. cDNA synthesis is carried out at 25°C for 10
minutes, followed by 120 minutes at 37°C. The reverse transcriptase is inactivated by heating at
85°C for 5 minutes, and the reaction is cooled to 4°C. The prepared cDNA is stored at 4°C if it is
to be used up within 24 hours, or cryopreserved if it is to be stored for a long time. The
composition of the reaction solution is as shown in Table 7.

12

CDC (2020) Research use only 2019-novel coronavirus (2019-nCoV) real-time RT-PCR primers and probes.
(https://www.cdc.gov/coronavirus/2019-ncov/lab/rt-pcr-panel-primer-probes.html)

17

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

Table 7 Preparation of Reverse Transcription Reaction Solution
Sample (RNA extract)10.0 μL
10× Reverse Transcription buffer

2.0 μL

25× dNTPs

0.8 μL

10× Random Primers

2.0 μL

MultiScribe™ Rverse Transcriptase (50 U/μL)

1.0 μL

RNase inhibitor (20 U/μL)

1.0 μLH2O

3.2 μL

Total

20.0 μL

Real-time PCR reaction
1. Make a 10-fold dilution of the positive control.
2. Prepare the reaction solution shown in Table 8 using the sample from which the reverse
transcription reaction was performed.
Table 8 Preparation of real-time PCR reaction solution
2×Master mix
12.5 μL
Forward primer (10 μM)
1.0 μL
Reverse primer (10 μM)
1.0 μL
TaqMan probe (10 μM)
0.75 μL
H2O
7.25 μL
Template (cDNA)
2.5 μL
Total
25.0 μL
3. Add 22.5 µL of the reaction solution to each well of a 96-well plate, with a minimum of two
wells per sample.
4. Add 2.5 μL each of positive control and sample cDNA. Add 2.5 μL H2O to the wells of thenegative control. In order to prevent contamination of the positive control, sealing or coveringwith aluminum foil is recommended.
5. Set the reaction conditions and start the reaction. However, since the optimal reaction
conditions vary depending on the real-time PCR system, reagents, and reaction vessel used,be sure to check the detection sensitivity, etc. in advance. Table 9 shows the reaction
conditions when the QuantiTect® Probe PCR Kit from QIAGEN is used as the reagent andthe Applied Biosystems 7500 or 7500 Fast real-time PCR system is used as the real-time PCRsystem.
Table 9 Example of reaction conditions for real-time PCR (Standard mode is selected)
95°C
15 min↓95°C
3 sec
×45 cycles
55°C
30 sec (Data collection)
6. The test is considered to be valid when the amplification curve of the positive control showsa rise and the amplification curve of the negative control does not show a rise.
7. To obtain the quantitative value of the viral gene copy number, construct a calibration curveusing the number of cycles obtained for the positive control dilution column. When thenormal logarithm of the positive control copy number is taken on the horizontal axis and thenumber of cycles on the vertical axis, the coefficient of determination of the calibration curve18

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

should be approximately 0.99 or higher, and the slope should be around -3.3. Whencalculating the amplification efficiency, it should be 90% or more. Confirm that quantifiability
is obtained when there are at least 10 copies per well (5 or more copies is more desirable).
8. If a rise in the amplification curve is observed in all wells of the same sample, the average
number of cycles obtained in each well shall be used as the number of cycles for that sample.
Even if the amplification curve rises in all wells of the same sample, if there is a differenceof one cycle or more between the wells, it is recommended that the real-time PCR be redone
for that sample.
9. When the amplification curve rises and does not rise among the wells of the same sample, it
is recommended that the real-time PCR be redone for that sample. If the results do not changeeven after redoing the real-time PCR, it is highly likely that the virus-derived gene is present
at a very low concentration in the sample, so the sample is judged to be merely positive
without calculating the copy number using a calibration curve, and the Ct value is reported asa reference value.
10. If the number of cycles of the sample is less than the number of cycles of the positive control
with the highest dilution ratio, the copy number should be calculated using a calibration curve.
If the number of cycles of the sample is greater than the number of cycles of the positive
control, the sample is judged to be merely positive without calculating the copy number, andthe Ct value is reported as a reference value.
11. It is recommended that the PCR plates and tubes containing the samples that tested positive
be stored frozen at -20°C or below, since sequencing analysis may be performed in the future
to confirm whether the PCR amplification was specific or not. When storing the samples, careshould be taken not to cause contamination by peeling off the caps or seals. Autoclaving ofplates after PCR should be avoided because of the possibility of contamination.

19

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

4. SAFETY MANAGEMENT IN SAMPLE HANDLING

Presence of infectious SARS-CoV-2 in wastewater has not been reported so far13.
Infectivity of SARS-CoV-2 is reportedly decayed faster than the viral RNA in wastewater14.
However, a suspicious case of COVID-19 transmission via wastewater route is also reported bya cohort study in unsanitary area15. Independent of presence of SARS-CoV-2, wastewater may
contain other pathogenic viruses and microbes, such as norovirus and toxigenic E. coli. To
avoid these potential risks, it is recommended to take the safety measures which is theappropriate to the actual situations of sample handling and surrounding situations of thelaboratory for analysis, as well as the epidemic situation of COVID-19 in the wastewater
catchment.
There is no legal standards nor regulations in Japan for the handling of wastewater samples
that may contain SARS-CoV-2 (as of December 2021). There is no legal standard for thehandling of sewage samples that may contain SARS-CoV-2 (as of February 2021). Hence, it isrecommended to follow the guidelines by relevant organization and industry associations if
available. For safety management in sample collection, WHO guidelines 13 on “Water,
sanitation, hygiene, and waste management for SARS-CoV-2, the virus that causes COVID-19”
would be helpful as a reference. In Japan, guidelines for infection prevention in sewage
operations16,17 are published by the Japan Sewer Collection System Maintenance Association
and Japan Sewage Facilities Management Association (Both guidelines are published inJapanese only).
Safety management is also recommended in sample handling for laboratory analysis
according to the actual situations of the analytical laboratory and the epidemic situation of
COVID-19 where the sample has been collected (Figure 2). Since the potential risk of SARSCoV-2 in wastewater is probably lower than clinical specimen, the required level of safety
management for handling of wastewater samples in a laboratory is equal to or lower than therequired level for clinical specimen. In the highest-level safety management equal to the clinicaltesting, a sample should be handled wearing personal protective equipment (PPE) in a Class II
biological safety cabinet. After analysis is completed, the samples and any containers andmicrotubes that may have been contaminated should be autoclaved before disposal. (reference,
WHO guidelines.) On the other hand, a sample can be handled as usual when the potential riskof SARS-CoV-2 in wastewater is considered to be negligible compared to those of otherpathogenic microorganisms in wastewater, regarding the epidemic situation of COVID-19 andthe actual situation of sample collection and laboratory. The following sections introduce
relevant guidelines from UNICEF/WHO and the National Institute of Infectious Diseases(NIID) as reference information for the development of safety control measures in laboratory
for SARS-CoV-2 RNA detection in wastewater.

13

WHO (2020) Water, sanitation, hygiene, and waste management for SARS-CoV-2, the virus thatcauses COVID-19. https://www.who.int/publications/i/item/WHO-2019-nCoV-IPC-WASH-2020.4
14Bivins, A., Greaves, J., Fischer, R., Yinda, K.C., Ahmed, W., Kitajima, M., Munster, V.J., Bibby, K.,
2020. Persistence of SARS-CoV-2 in Water and Wastewater. Environ. Sci. Technol. Lett. 7, 937–942.
15
Yuan et al. (2020). Sewage as a possible transmission vehicle during a coronavirus disease 2019
outbreak in a densely populated community: Guangzhou, China, April 2020. Clinical Infectious Diseases,
April, 1–14.
16
Japan Sewer Collection System Maintenance Association (2021) Guidelines for countermeasuresagainst novel coronavirus infection in sewerage pipeline management operations (in Japanese).
https://www.jascoma.com/topics/2020/coronavirus_disease/images/20210914/information_1.pdf
17Japan Sewage Facilities Management Association (2021) Guidelines for Prevention of Novel
Coronavirus Infection in Sewage Facility Operation and Management (in Japanese) https://www.gesuikanrikyo.or.jp/pdf/news_2021102901.pdf

20

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

Figure 2 Approach to determine safety management in handling wastewater samples that may
contain SARS-CoV-2. It is suggested to take flexible safety management measures according to
the situation of the sampling site, COVID-19 epidemic in the target community, and theguidelines of related organizations.
4.1 Handling of wastewater samples
The purpose of personal protective equipment (PPE) is to prevent exposure to wastewater
aerosols and to prevent its intrusion into uncontaminated areas. It is important to take off the
PPE before entering the uncontaminated area. The appropriate PPE should be selectedaccording to the potential risk of infection based on the epidemic situation of COVID-19 in thewastewater catchment and the location of the sampling site. In general, higher risk is expected
when more infections are present in the target population and/or when the sampling site is closer
to the epidemic sources. The potential risk of treated wastewater is lower than that of untreatedwastewater.
For handling of untreated wastewater under COVID-19 outbreak situations, Japan Sewer
Collection System Maintenance Association and Japan Sewage Facilities Management
Association recommend the following safety management in their guidelines.
Ø Wearing of masks and gloves is recommended during on-site work.
Ø When performing work that may involve direct contact with untreated wastewater, ye
protection wear (e.g. safety goggles) is encouraged to prevent direct exposure to splashon their eyes. Wearing the personal protective equipment (e.g. face shield andprotective clothing) is encouraged upon necessity.
Ø Clothing and equipment that have been contacted untreated wastewater should be
washed and disinfected.
The UNICEF/WHO Interim Guidance on Water, sanitation, hygiene, and waste
management for SARS-CoV-2, the virus that causes COVID-19 (July 29, 2020) recommends
the following
For personnel working with untreated sewage for which there are considerable infectiousrisks, standard PPE should be worn (protective outerwear, heavy-duty gloves, boots,masks, goggles or a face shield). It should be worn at all times when handling ortransporting excreta offsite, and great care should be taken to avoid the splashing andrelease of droplets. For sanitation workers, this includes pumping out tanks or unloading
pumper trucks. After handling the waste and once there is no risk of further exposure,21

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

individuals should safely remove their PPE and perform hand hygiene before entering
the transport vehicle. Soiled PPE should be put in a sealed bag for safe laundering later(see Environmental cleaning and laundry). Workers should be properly trained in how to
put on and remove PPE, so that these protective barriers are not breached. If PPE is notavailable or the PPE supply is limited, the frequency of correct hand hygiene should
increase, and workers should keep at least 1m distance from suspected or confirmed
cases.
The guideline also recommends the adequate manner for hand disinfections after contact
with samples at potential risk for COVID-19. It recommended to perform hand disinfection
before and after wearing PPE (personal protective equipment); e.g. when changing gloves, after
contact with the environment around untreated wastewater. It also advise to practice disinfectionof shoes by using a disinfectant mat, if possible.
If hands are not visibly dirty, the preferred method is using an
alcohol-based hand rub for 20−30 seconds using the appropriatetechnique. When hands are visibly dirty, they should be washed withsoap and water for 40−60 seconds using the appropriate technique.

Wastewater samples spilled during the experiment in a laboratory should be wiped off withpaper towel after spraying sufficient amount of disinfection ethanol. To ensure the disinfection,
5% bleach solution can be also used. Sample containers that may have contacted with thesample (including paper towels used to wipe off the sample) should be autoclaved before
disposal.
4.2 Safety management in laboratory work for wastewater analysis
When the wastewater sample has potentially high risk from the situation of sampling site
and COVID-19 outbreak, the highest level of safety is ensured by applying the safety
management as strict as clinical specimen for COVID-19 testing. For the handling of medical
specimens that may contain SARS-CoV-2, the provisional rules developed by the National
Institute of Infectious Diseases (NIID) are widely used in Japan. (Note that this guideline wasdeveloped for handling of clinical specimen including nasopharyngeal swab samples, not for
wastewater samples. Since the potential risk of wastewater is smaller than that of clinicalspecimens, handling of wastewater samples does not require more stringent safety control than
this guideline.)
The National Institute of Infectious Diseases (NIID) has established the following internal
rules (dated February 21, 2020) 18.
1. The pathogen of novel coronavirus 2019-nCoV will be handled as BSL3/ABSL3.
2. Clinical specimens derived from patients suspected infection with novel coronavirus
2019-nCoV will be handled as BSL2.
Accordingly, the 2019-nCoV Detection Manual (Ver. 2.9)19 by the National Institute of
Infectious Diseases (NIID) describes about operational precautions as below.
Specimens should be handled ... in a biosafety cabinet in the BSL2 experimental facility.
Personal protective equipment (PPE) such as disposable gowns, gloves (double), masks

18

National Institute of Infectious Diseases (2020) Handling of the new coronavirus SARS-CoV-2 in the
National Institute of Infectious Diseases. https://www.niid.go.jp/niid/ja/byougen-kanri/9367-n-covbio.html
19
National Institute of Infectious Diseases (2020) 2019-nCoV Detection Manual (Ver. 2.9)https://www.niid.go.jp/niid/images/lab-manual/2019-nCoV20200318v2.pdf

22

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER(surgical masks are acceptable), and caps should be worn during sample handling. Before
opening the microtube lid, centrifuge first and use a tube opener to prevent the generationof aerosols.
The National Institute of Infectious Diseases (NIID) sample handling precautions are inaccordance with the WHO post-outbreak biosafety guidelines for handling of SARS-CoV
specimens and cultures (December 18, 2003)20. The guideline stipulates about handling ofspecimens suspected of containing SARS coronavirus as below.
<Handlings requiring infection prevention measures>
・ Aliquoting and/or diluting specimens
・ Inoculation of bacterial or mycological culture media
・ Performance of diagnostic tests that do not involve propaganda of viral agents in vitro orin vivo・ Nucleic acid extraction procedures involving untreated specimens
・ Preparation and chemical- or heat-fixing of smears for microscopic analysis<Infection prevention measures to be implemented>
・ Any procedure that may generate aerosols or droplets should be performed in abiological safety cabinet (e.g., sonication, vortexing).
・ Laboratory workers should wear protective equipment, including disposable gloves,solid-front or wrap-around gowns, scrub suits, or coveralls with sleeves that fully cover
the forearms, head covering and, where appropriate, shoe covers or dedicated shoes, eyeprotection and a surgical mask, or full-face shield, because of the risk of creating
aerosols or droplets exposure when performing specific manipulations.
・ Centrifugation of specimens should be performed using sealed centrifuge rotors or
sample cups. These rotors or cups should be unloaded in a biological safety cabinet.
・ Work surfaces and equipment should be decontaminated after specimens are processed.
Standard decontamination agents that are effective against enveloped viruses should be
sufficient if used according to the manufacturer’s recommendations. Generally, 5%
bleach solutions are appropriate for dealing with biohazardous spillage. Moreinformation on disinfection and sterilization is provided in the WHO Laboratory
Biosafety Manual, 3rd revised edition.
・ Biological waste contaminated with suspect or confirmed SARS specimens, or with
SARS-CoV, should be treated as outlined in the WHO Laboratory Biosafety Manual,
3rd revised edition before disposal.
・ When a procedure or process cannot be conducted within a biological safety cabinet,
then appropriate combinations of personal protective equipment (e.g. respirators, faceshields) and physical containment devices (e.g. centrifuge safety cups or sealed rotors)
must be used.
Clinical specimen laboratories are stipulated to be sterilized and disposed as soon as
possible after the pathogen is detected. In the case of wastewater samples, it is recommended toautoclave and dispose the sample after SARS-CoV-2 is detected if it is not planned to be usedfurther.

WHO (2003) post-outbreak biosafety guidelines for handling of SARS-CoV specimens andcultures. https://www.who.int/publications/m/item/who-post-outbreak-biosafety-guidelines-forhandling-of-sars-cov-specimens-and-cultures
20

23

DETECTION MANUAL FOR SARS-CoV-2 RNA IN WASTEWATER

4.3 Other reference information on pathogen management○ Ministry of Health, Labour and Welfare: Regulations for the Control of Specified
Pathogens, etc. under the Infectious Diseases Control Law
https://www.mhlw.go.jp/stf/seisakunitsuite/bunya/kenkou_iryou/kekkakukansenshou17/03.html○ National Institute of Infectious Diseases: Packaging containers for transporting pathogens Basic triple packaging configuration
https://www.niid.go.jp/niid/ja/from-biosafe/947-youkisb.html

24