WaterRA ColoSSoS Project

Inter-laboratory Study: SARS-CoV-2 in Wastewater

Dr Kate Griffiths

Bioanalysis Section, Australian National Measurement Institute

14th June 2021

Measurement for a fair, safe, healthy and competitive Australia | measurement.gov.au

1
Wastewater surveillance for SARS-CoV-2

• Phase 1: Method development

• Multiple labs developed methods independently, using a range of materials for validation and calibration

• Recovery methods: electronegative membranes, ultrafiltration, PEG, ultracentrifugation

• RNA extraction methods: range of kits available

• RT-qPCR kits, mastermixes, 1-step or 2-step and assays

• Choice of calibrant / reporting units

• Phase 2: Compare method performance between different service providers

• Limit of detection (LOD) / yield

• Reproducibility

• Quantitative accuracy

• Participants:

• 11 in Australia and 1 in New Zealand

• 2 water utilities, 3 Government labs, 2 commercial labs, 5 University labs;

2
Plan for the ColoSSoS inter-laboratory study

• Study materials provided:

– 10 x 50 mL aliquots of domestic wastewater (grab sample, untreated)

– 2 x frozen aliquots of inactivated virus (‘V1’ and ‘V2’) for spiking

– 3 x sets of calibrant CRM (6 dilutions prepared from inactivated virus)

We were very fortunate to have domestic wastewater expected to be free of the virus and gamma-irradiated 

SARS-CoV-2 provided by the Victorian Infectious Diseases Reference Laboratory (VIDRL) – both game changers.

3
NMI Calibrant CRM

• The NMI calibrant: 

• Prepared from gamma-irradiated SARS-CoV-2 virus supplied by VIDRL

• 6-point dilution series: 560, 245, 62, 18.5, 6.5 and 2.1 copies per 5 μL

• Quantified in “copy number concentration of SARS-CoV-2 genome equivalents”

• Quantified using RT-dPCR

• Measurement uncertainty includes reverse transcription 

efficiency for conversion of RNA to DNA, using isotope 

dilution mass spectrometry (IDMS) data

• Provides traceability to the International System of Units

(SI mole)

• 5 μL of each dilution to be added directly into the RT-PCR well

4
Analysed straight away = Day 0

RNA samples analyzed in duplicate

and at two concentrations

Stored at 4°C for 2 days prior to analysis = Day 2

RNA samples analyzed in duplicate

and at two concentrations

100 μL V1 = 740,000 *cp 100 μL V2 = 66,000 *cp

S1 S2 S10

(NEC2)

S9

(NEC1)

S3 S4 S5 S6 S7 S8

Study Instructions

5

Calibrant (6 dilutions) analyzed in duplicate, plus NECs and NTCs

*copies of 

SARS-CoV-2 

genome equivalents
Results

spreadsheet

Please fill in all the cells coloured yellow

Concentration Factor Vol. before processing (mL) 50 CF (Dilution 1)

Vol. after virus recovery (mL) CF (Dilution 2)

Vol. used in RNA extraction (mL)

Vol. of eluted RNA (mL)

RNA Dilution Factor 1 

RNA Dilution Factor 2

First Day, Assay 1 First Day, Assay 2

Calibrant 

Number

Replicate 

Number

Copy Number 

per RT-qPCR

Log (Copy 

Number) Cq Value Calibrant Number

Replicate 

Number

Copy Number 

per RT-PCR

Log (Copy 

Number) Cq Value

C1.1 580 2.76 #N/A NA050 C1.1 580 2.76 #N/A

C1.2 580 2.76 #N/A C1.2 580 2.76 #N/A

C2.1 245 2.39 #N/A NA051 C2.1 245 2.39 #N/A

C2.2 245 2.39 #N/A C2.2 245 2.39 #N/A

C3.1 62.0 1.79 #N/A NA052 C3.1 62.0 1.79 #N/A

C3.2 62.0 1.79 #N/A C3.2 62.0 1.79 #N/A

C4.1 18.5 1.27 #N/A NA053 C4.1 18.5 1.27 #N/A

C4.2 18.5 1.27 #N/A C4.2 18.5 1.27 #N/A

C5.1 6.5 0.81 #N/A NA054 C5.1 6.5 0.81 #N/A

C5.2 6.5 0.81 #N/A C5.2 6.5 0.81 #N/A

C6.1 2.1 0.32 #N/A NA055 C6.1 2.1 0.32 #N/A

C6.2 2.1 0.32 #N/A C6.2 2.1 0.32 #N/A

Sample 

Number

Replicate 

Number

Cq Value Calculated log 

value

Estimated 

Copy 

Number/

RT-qPCR

Mean Estimated 

Copy Number/

RT-qPCR

Sample 

Number

Replicate 

Number

Cq Value Calculated log 

value

Estimated 

Copy 

Number/

RT-qPCR

Mean Estimated 

Copy Number/

RT-qPCR

S1.1 S1.1

S1.2 S1.2

S1.3 S1.3

S1.4 S1.4

S2.1 S2.1

S2.2 S2.2

S2.3 S2.3

S2.4 S2.4

S5.1 S5.1

S5.2 S5.2

S5.3 S5.3

S5.4 S5.4

S6.1 S6.1

S6.2 S6.2

S6.3 S6.3

S6.4 S6.4

S9.1 S9.1

S9.2 S9.2

NTC1 NTC1

NTC2 NTC2

Assay 1 Assay 2

Slope (m)

Intercept

Correlation coefficient (r2)

Efficiency

Number of points on calibration curve

Negative

Controls

Sample 9

(NEC)

Negative

Controls

Samples

Sample 1

Sample 2

Sample 9

(NEC)

NTC

Sample 5

Sample 6

Sample 5

NTC

Samples

Sample 1

Sample 6

Sample 2

Laboratory Name:

Analyst Name:

Analysis date (first day)

V1 Spike aliquot number:

V2 Spike aliquot number:

CalibrantCalibrant

NA050

NA051

NA052

NA053

NA054

NA055

20

25

30

35

40

45

0.00 0.50 1.00 1.50 2.00 2.50 3.00

Cq

Log10 concentration

Assay 2, First Day

20

25

30

35

40

45

0.00 0.50 1.00 1.50 2.00 2.50 3.00

Cq

Log10 concentration

Assay 1, First Day

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Analysis of calibrant data

Lab 6, US CDC N1 Assay

Slope of -3.32 = 100 % RT-qPCR efficiency

Second Day, Assay 1

Calibrant 

Number

Replicate 

Number

Copy Number 

per RT-qPCR

Log (Copy 

Number) Cq Value

C1.3 580 2.76 26.84

C1.4 580 2.76 26.79

C2.3 245 2.39 28.02

C2.4 245 2.39 28.03

C3.3 62.0 1.79 30.04

C3.4 62.0 1.79 30.00

C4.3 18.5 1.27 31.94

C4.4 18.5 1.27 31.91

C5.3 6.5 0.81 33.16

C5.4 6.5 0.81 33.42

C6.3 2.1 0.32 34.47

C6.4 2.1 0.32 35.25

Calibrant

NA050

NA051

NA052

NA053

NA054

NA055

7

y = -3.3196x + 35.994

R2 = 0.996

20

25

30

35

40

45

0.0 1.0 2.0 3.0

C

q

Log10 concentration

Slope

C Intercept q 

log10ci 
Reasons for using a calibrant

1. Measure reaction efficiencies

2. Report in appropriate units:

Copy number concentration of SARS-CoV-2 genome equivalents

Reported Cq values for the highest calibrant dilution range from 25.31 to 33.77

Reporting in Cq values, a ΔCq of 8.43 represents 345x difference in copy number concentration.

In reality this is the same material measured by 12 different labs using 4 different RT-PCR assays

Average Cq values produced by all participants using most 

concentrated calibrant dilution (NA050) @ 580 copies/5 μL ±20 %.

8

22

24

26

28

30

32

34

China CDC N

gene

China CDC

ORF1ab

US CDC N1

gene

US CDC N2

gene

C

q

valu

es for calibrant dilutio

n

N

A

0

5

0
Reasons for using a calibrant

1. Measure reaction efficiencies

2. Report in appropriate units

3. Reduced assay-specific bias

Assay biases: 

• amplicon length

• secondary structures

• fluorescence chemistries

• threshold position

9

Average Cq values produced by all participants using most 

concentrated calibrant dilution (NA050) @ 580 copies/5 μL ±20 %.

22

24

26

28

30

32

34

China CDC N

gene

China CDC

ORF1ab

US CDC N1

gene

US CDC N2

gene

C

q

valu

es for calibrant dilutio

n

N

A

0

5

0

It’s preferable to report in traceable 

units rather than Cq values
0.1

1.0

10.0

100.0

1000.0

10000.0

7 13 2 10 3 4 6 12 1 8 5 9 #2 9 #1

R

N

A

c

o

pies/

m

L

w

aste

w

ater (lo

g sc

ale)

Laboratory Number

Reasons for using a calibrant

1. Measure reaction efficiencies

2. Report in appropriate units

3. Reduced assay-specific biases

4. Provides traceability, allowing direct comparison 

of data between different laboratories

Calibrant → copies per μL of extracted RNA

Concentrafion Factor (CF) → copies per mL of wastewater

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Concentration Factor (CF)

Reference: Pecson, B. M. et al. (2021) “Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw 

wastewater: findings from an interlaboratory methods evaluation in the US”. Environmental Science Water Research and Technology 7: 504-520.

CF

Allows conversion of copies per μL of extracted RNA into copies per mL of wastewater

11
Wastewater samples S1 and S2: spiked with ‘V1’, analyzed on Day 0

Labs 5, 6 and 13 only used one assay; labs 2, 8 and 12 had data excluded for one assay

12

1

10

100

1000

1 2 3 4 5 6 7 8 9 #1 9 #2 10 12 13

R

N

A

c

o

pies/

m

L

of w

aste

w

ater

(lo

g sc

ale)

Laboratory Number

Assay 1 V1 diluted Assay 2 V1 diluted
Summary of protocols used by participants

Virus Recovery RNA Extraction RT-PCR

Eight labs used electronegative 

membranes with range of pretreatments: 

• centrifugation,

• acidification,

• addition of MgCl2

One lab used 20 % PEG

Two lab used ultrafiltration:

• centrifugal filters

• hollow-fibre concentrating 

pipette

Seven labs used Qiagen kits:

• PowerSoil

• PowerWater

• PowerMicrobiome

• PowerViral

Three labs used ThermoFisher kits:

• MagMax Viral Pathogen

• MagMax Microbiome Ultra

Other kits used:

• Macherey-Nagel RNA stool kit

• Roche High Pure Viral Nucleic 

Acid

• Vazyme fast pure viral RNA/DNA

Five labs used PerkinElmer kit 

including China CDC assays

Two labs used Promega kit 

including US CDC assays

Other kits used:

• ThermoFisher Combo kit,

• Vazyme kit,

Three labs bought primers, probes, 

enzymes and buffers separately, 

including one using 2-step RT-PCR.

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0.0%

0.5%

1.0%

1.5%

2.0%

2.5%

3.0%

3.5%

4.0%

13 7 4 6 3 10 5 1

%

R

e

c

o

v

e

r

y

f

r

o

m

V

1

Laboratory Number

Recoveries

Percentage of recoveries of ‘V1’ spiked samples, assuming 740,000 SARS-CoV-2 genome equivalents were spiked into the 50 mL of 

wastewater (14,800 copies per mL of wastewater).

Error bars are expanded relative uncertainties (95%) capturing participant measurement variation and V1 measurement uncertainty.

x 100 Total viral RNA genome equivalents recovered

Total viral RNA genome equivalents added

• Based on ‘V1’ spike = quantified in genome 

equivalents after gamma-irradiation, freezethawing and lysing.

• Yields may underestimate recovery efficiency 

for whole virus particles.

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Detailed comparison of protocols with highest recoveries

Lab 7 Lab 13

Pre-treatment

Centrifugation @3,270 g spin at 4 °C for 30 min

(solids removal)

Centrifugation @10,000 g for 30 min with extract from 

solids added back to cleared supernatant

pH Adjustment pH 3.5 pH 7.0-7.2

Virus recovery 0.45 μm electronegative membrane, vacuum pump 20 % PEG precipitation, 2 hr incubation at 4 °C,

10,000 g spin for 30 min 

RNA extraction RNeasy PowerMicrobiome® Kit (Qiagen)

ShieldTM reagent and phenol added during bead beating High Pure Viral Nucleic Acid kit (Roche)

Elution/diluent Nuclease-free water Nuclease-free water

Style of RT-PCR 1-step 2-steps

Assays China CDC assays: N gene (FAM) and ORF1ab (Texas Red) China CDC assay: N gene, FAM-labelled probe

Temp. during plate set-up 25 °C 4 °C

Enzymes/mastermixes SARS-COV-2 Real Time 1-step RT-PCR assay kit (PerkinElmer) SuperScript III® Reverse Transcriptase (Thermofisher) 

PerfeCta® qPCR ToughMix (Quanta Biosciences)

Cycling parameters

RT: 37°C, 2 min, 50°C, 5 min, 42°C, 35 min

Enzyme Activation: 94°C, 10 min

Denaturation: 94°C, 10 sec

Annealing: 55°C, 15 sec, Extension: 65°C, 45 sec

Number of Cycles: 45

RT: 50°C, 30 min

Enzyme Activation: 95°C, 180 sec

Denaturation: 95°C, 15 sec

Annealing/Extension: 60°C, 30 sec

Number of Cycles: 45

Dilution(s) for RNA Neat and 1-in-10 dilution Neat and 1-in-4 dilution

Concentration Factors CF1: 323, CF2: 32 CF1: 200, CF2: 50

15
Conclusions

• All participants successfully detected RNA from the inactivated V1 virus spiked 

into wastewater

• Highest recoveries, great reproducibility and clean RNA using:

• electronegative membranes with centrifugation and acidification as pretreatments

• 20 % PEG

• Major implications for lower income regions and ColoSSoS DFAT program

• PEG method widely used for environmental surveillance of poliovirus – familiar and low cost*

• SARS-CoV-2 calibrant and Concentration Factor for comparable reporting units:

“RNA copy number concentration of SARS-CoV-2 genome equivalents 

per mL of wastewater”

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*World Health Organization (2003), Guidelines for environmental surveillance of poliovirus circulation, WHO/V&B/03.03 
Acknowledgements

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• WaterRA for supporting and co-ordinating this study:

• Dr Kelly Hill and Marlene Hsu

• Victorian Infections Diseases Reference Laboratory for providing gamma-irradiated SARS-CoV-2:

• Prof. Bruce Thorley and Dr. Julian Druce

• Task Leader for the ColoSSoS Task Group 2 and ColoSSoS Project Manager:

• Dr Dan Deere

• All of the study participants

• Sydney Water for providing the domestic wastewater sample:

• Kate McLennan and Anna Flack

• Pathwest and Sydney Water Laboratories for unwittingly assisting with the validation of the V1 

and V2 material:

• Jake Gazeley and Sudhi Payyappat

• The NMI Bioanalysis Team:

• Dr D.G. Burke, (Manager); Dr K.R. Emslie (NMI Honorary Fellow); Dr A. Baoutina; Dr S. Bhat; Dr M. Forbes-Smith; 

Ms F. Hall; Dr D. Lynch; Mr J. McLaughlin; Ms L. Partis; Dr L.B. Pinheiro
Contact us for inquiries and calibrant CRM sales

Dr Kate Griffiths

National Measurement Institute

Email: BioRM@measurement.gov.au

Phone: +61 2 8467 3730

36 Bradfield Road, West Lindfield

Sydney, NSW 2070, Australia

measurement.gov.au

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