TITLE: Quantification of SARS-CoV-2 in wastewater
AUTHORS: David Ian Walker, James Lowther, Nick Evens, Jonathan Warren, Jonathan Porter, Kata Farkas, Davey Jones

[DESCRIPTION]
This procedure describes the concentration and quantification of SARS-CoV-2 from wastewater as used by the Environmental Monitoring for Health Protection (EMHP) in England programme during the COVID-19 pandemic. It is based on the standard operating procedure developed by a collaboration between Environment Agency National Laboratory Service and Bangor University. This generic protocol was prepared by Cefas to allow laboratories to carry out SARS-CoV-2 quantification from wastewater samples in a way that is similar to the EA’s procedure, while still allowing some variation where necessary.

Viral concentration is by ammonium sulphate precipitation followed by centrifugation. Viral RNA extraction is by lysis with guanidine isothiocyanate (GITC) and adsorption to silica. Extracted viral RNA is amplified and detected by quantitative RT-PCR (RT-qPCR). This protocol describes a method for quantification of virus RNA in the test sample and includes production of control materials.

The wastewater samples are first clarified by centrifugation. A precipitant (ammonium sulphate) is then added before the sample is incubated for 1 hour at 4°C. This is followed by centrifugation to generate a viral pellet that is suitable for viral nucleic acid extraction. Details of addition of a spike process control (phi6) to the test samples are also described.

It is necessary to extract RNA using a method that yields clean RNA preparations to reduce the effect of PCR inhibitors. In this protocol the chaotropic agent GITC is used to disrupt the viral capsid. RNA is then adsorbed to silica to assist purification through several washing stages. Purified viral RNA is released from the silica into a buffer prior to RT-qPCR. The use of the Thermofisher KingFisher Flex Purification System with BioMerieux’s NucliSENS extraction reagents is described here. Other RNA extraction methods that use GITC followed by silica-based purification may also be applicable if they can be demonstrated to give equivalent results.

This protocol uses one-step RT-qPCR using hydrolysis probes. In one-step RT-qPCR, reverse transcription and PCR amplification are carried out consecutively in the same tube. Hydrolysis probe-based qPCR utilises a short DNA probe with a fluorescent label and a fluorescence quencher attached at opposite ends. The assay chemistry ensures that as the quantity of amplified product increases, the probe is broken down, and the fluorescent signal from the label increases proportionately. Fluorescence may be measured at each stage throughout the cycle. The first point in the PCR cycle at which amplification can be detected for any reaction is proportional to the quantity of template, therefore analysis of the fluorescence plots enables determination of the quantity of target sequence in the sample.


[GUIDELINES]
RT-qPCR 

The instructions below are for use with the EA’s RT-qPCR master mixes for the N1 and phi6 assays. Alternative RT-qPCR master mixes and cycling conditions may also be used if they follow the general requirements listed in “General requirements for RT-qPCR master mixes” (page 5) and below and have been demonstrated to give equivalent results.

General requirements for RT-qPCR set-up

RT-qPCR should be carried out using the primers and probes outlined in “RT-qPCR PCR master mixes”.

Full sets of RT-qPCR controls and quantification standards should be used for every plate where sample RNA is assayed for that RT-qPCR target. For example, if a single RT-qPCR plate is to be used to analyse both SARS-CoV-2 and phi6 simultaneously, no template control (NTC) and ssRNA standard dilutions will be needed for both targets on the same plate.
Prepare RT-qPCR master mixes immediately before starting procedure.
Sample RNA and ssRNA quantification standards should be kept separate. It is preferable that they are added to the RT-qPCR plate in separate laboratories to avoid the potential for contamination of samples by ssRNA quantification standard. However, it is also possible to maintain separation of sample RNA and ssRNA quantification standards by using automated liquid handling systems. Laboratories must ensure that local quality procedures are maintained to reduce the possibility of cross contamination of this sort.

Analysis of results

Quality of amplification data

Analyse the amplification plots using the approach recommended by the manufacturer of the realtime PCR machine. The threshold should ideally be set so that it crosses the area where the amplification plots (logarithmic view) are parallel (the exponential phase).

Check all amplification plots to identify false positive results caused by high or uneven background signal (Figure 1)

 
Results for any wells affected in this way should be regarded as negative.

Check all amplification plots to identify true positive plots where the recorded quantification cycle (Cq) value is significantly distorted by high or uneven background signal. Approximate correct Cq values should be noted (in addition to the recorded value) for any wells affected in this way. Corrected Cq values should be used for all quantity calculations. Figure 2 shows a case where the recorded Cq value was 15.5, however it should be noted by the analyst that the correct figure should be e.g. 34.

 
Status of controls

No template controls (NTC)

Expected result: No amplification of N1, no amplification of phi6

Explanation: A positive result for the NTC indicates background contamination of the RT-qPCR.

Outcome of unexpected result: Repeat the entire RT-qPCR plate. The RT-qPCR set-up environment should be decontaminated. Also consider replacing PCR plasticware, TEX buffer, primer and probe working aliquots and RT-qPCR master mix. If contamination of NTC continues following multiple RTqPCR repeats, investigate potential contamination sources and resolve as needed.

Total blank control

Expected result: No amplification of N1, no amplification of phi6

Explanation: A positive result for the total blank control indicates that there was contamination of the control at some point within the sample preparation process. This may have been due to contamination of the workstations where the samples were processed or by cross-contamination between samples.

Outcome of unexpected result: Repeat the RT-qPCR to confirm contamination. If no contamination is seen after repeating the RT-qPCR, then repeat RT-qPCRs for associated samples. If contamination of the total blank control is confirmed, all sample results associated with that control should be rejected.

Construction of standard curves

Calculate the r² (coefficient of determination), slope and intercept values for a plot of the Cq values of the ssRNA standard dilutions against log10 concentrations (copy numbers/µl).

Curves with r² <0.98 and the slope not between -3.6 and -3.1 (equivalent of 90% to 110% amplification efficiency) should not be used to calculate results.

If r² or slope fall outside these values, then up to two anomalous results may be removed from the standard curve and the r², slope and intercept values recalculated.

If r² or slope do not fall within these values after removal of anomalous results, then all RT-qPCR results should be rejected for that plate and the RT-qPCRs repeated for the associated sample RNAs.

Calculation of phi6 recovery

Estimate the recovery of the phi6 process control by comparing the Cq value for phi6 for each sample RNA reaction against the Cq value for the phi6 positive control as follows:

Phi6 process control recovery = 10(Δ𝐶𝐶𝑞𝑞/m) × 100%

where ΔCq = Cq value (sample RNA) – Cq value (phi6 positive control virus RNA) and m = slope of the phi6 RNA standard curve.

For each sample, calculate the % recovery separately for each replicate then calculate an average (arithmetic mean) of all replicate recovery values to determine an overall recovery for the sample.

Where recovery is <0.01%, the results are not valid and the sample must be retested.

Calculation of gc/µl (G)

To calculate the gene copies per reaction (gc/µl), use the following equation:

𝐺 = 10(Δ𝐶𝑞/m)

where G =gc/µl, ΔCq = Cq value (sample RNA) – standard curve intercept and m = slope of the ssRNA standard curve.

For example:

For a sample RT-qPCR run where the standard curve slope was -3.37 and the intercept was 35.6, for a sample RT-qPCR giving a result of Cq value of 30.5, the gc/µl would be calculated as:

Δ𝐶𝑞 = 30.5 − 35.6

= −5.1

and so

G = 10(−5.1/−3.37)

= 32.6 gene copies/µ𝑙

RT-qPCR replicates with negative results should be given a concentration of 0 gc/µl. 

Determining variability between replicates (ΔG)

Before using G to calculate gene copies per liter, determine whether the duplicate reactions fall within acceptable levels of variability i.e. if both replicate results are below limit of quantification (LOQ) or if for both replicates G differs from the mean of G by <50% of the mean of G.

LOQ and limit of detection (LOD) for the RT-qPCR must be calculated experimentally in your laboratory in line with guidance from the reference laboratory. In the absence of an experimentally determined LOQ, a nominal LOQ of 4 gc/µl should be used in the short-term. In the absence of an experimentally determined LOD, a nominal LOQ of 0.4 gc/µl should be used in the short-term. However, LOQ and LOD must be calculated as quickly as possible to ensure that results accurately reflect the limitations of your procedure.

If both replicates G differs from the mean of G by <50% of the mean of G, then the difference between replicate results in ≤3 times the lowest replicate. Therefore, this quality control parameter can be calculated using the following equation:


 
If GP is ≤3, then the variability between replicates is acceptable. Examples of acceptable and unacceptable results are shown in Table 6.

Table 5: Examples of acceptable and unacceptable replicate results 
 
Calculate gene copies per litre

Calculate the arithmetic mean average gc/µl for the duplicate RT-qPCR reactions (GM).

The sample concentration in gene copies per litre (gc/l) should be calculated using the following equation:

 
where GL = sample concentration (gc/l), Ve = RNA elution volume (µl), VS = volume of wastewater processed (ml).

For example:

Assuming an elution volume of 120 µl, where 150 ml of wastewater was processed. For a sample with duplicate G values of 32.6 gc/µl and 29.4 gc/µl, the sample concentration would be calculated as:


 
LOQ and LOD for the RT-qPCR must be calculated experimentally in your laboratory. In the absence of an experimentally determined LOQ, where a nominal LOQ of 4 gc/µl is applied, the LOQ for the whole process is 3,200 gc/l. In the absence of an experimentally determined LOD, where a nominal LOD of 0.4 gc/µl is applied, the LOD for the whole process is 160 gc/l. However, LOQ and LOD must be calculated as quickly as possible in line with guidance from the reference laboratory.

Summary of rejection and retesting criteria

Positive result in NTC: Repeat RT-qPCR plate
Positive result in RNA extraction negative control: Repeat RT-qPCR. Repeat RNA extractions if possible and if contamination is still present. Reject all sample results associated with RNA extraction negative control if is contamination still present.
Positive result in total blank control: Repeat RT-qPCR. Reject all sample results associated with total blank control if is contamination still present.
Quantification standard curve slope >-3.1 or <-3.7 after outliers removed: Repeat RT-qPCR plate
Quantification standard curve r² <0.98 after outliers removed: Repeat RT-qPCR plate
Recovery <0.01%. Repeat RNA extraction if possible. Reject sample result if recovery remains <0.01%.
At least one RT-qPCR duplicate is >LOQ and ΔG is ≥50% of GM: Repeat RT-qPCRs for that sample.

Reporting results

The following information from the RT-qPCR analyses must be reported for each sample along with the rest of the sample information and data (e.g. sample metadata and inorganics data).

RT-qPCR run end time and date
Wastewater sample volume (150 ml)
Name of the SARS-CoV-2 target (N1)
Volume of template RNA added to RT-qPCR reactions
Gene copies per µl of template RNA for each RT-qPCR replicate
o For samples with no result, this should be left blank
Mean gene copies per µl of template RNA
o For samples with no result, this should be left blank
Cq for each sample RT-qPCR reaction
o Negative Cq values should be reported as 0.00
o For samples with no result, this should be left blank
SARS-CoV-2 concentration in sample (gc/l)
o For samples with no result, this should be left blank
Limit of detection (LOD) for SARS-CoV-2 (gc/l)
Limit of quantification (LOQ) for SARS-CoV-2 (gc/l)


 
 
 

[STEPS]
SECTION: Preparation of daily phi6 process control working suspensions
1. On each day of testing, a new batch of phi6 process control working suspension should be made.
SECTION: Preparation of daily phi6 process control working suspensions
2. Remove an aliquot of phi6 stock from the freezer (prepared according to Appendix 1), thaw and mix thoroughly by vortexing.
SECTION: Preparation of daily phi6 process control working suspensions
3. Create a working suspension of phi6 by adding phi6 stock to Ringer’s ¼ Strength Solution to a final volume of 50±0.5 ml and mixing thoroughly by inverting several times.
 

SECTION: Initial sample clarification
4. Mix each sample by inverting the sample bottle 10 times within a biological safety cabinet.
SECTION: Initial sample clarification
5. Measure approximately 200 mL of sample into a suitable centrifuge bottle.

SECTION: Initial sample clarification
6. Prepare at least two control centrifuge bottles with approximately 200 mL of spring water on each day of testing.

SECTION: Initial sample clarification
8. Place the centrifuge rotor into a biological safety cabinet and carefully remove the samples from the rotor being careful not to disturb the pelleted solids.
 

SECTION: Virus concentration and lysis
9. On a digital balance, carefully decant 150±1 g of the clarified supernatant into clean centrifuge bottles, being careful to minimise the amount of pelleted solids that are transferred.
SECTION: Virus concentration and lysis
10. To each 150 mL clarified sample and one of spring water control samples, add 1±0.01 ml of phi6 process control. Mix the samples well by shaking or inverting several times.

SECTION: Virus concentration and lysis
11. Pour the sample/phi6 mixtures in to centrifuge bottles containing 60±1 g of ammonium sulphate.
 

SECTION: Virus concentration and lysis
12. Dissolve the ammonium sulphate by inverting the bottles several times, or by shaking on an orbital shaker at approximately 200 rpm until all of the ammonium sulphate has dissolved.

SECTION: Virus concentration and lysis
13. Incubate the samples at 3±2°C for at least 60 to 180 minutes.
SECTION: Virus concentration and lysis
14. Mark the outer sides of the bottle where the pellets will form during centrifugation.
SECTION: Virus concentration and lysis
15. Centrifuge the chilled samples for 30 min at 10000 x g at 4±1°C with no braking.

SECTION: Virus concentration and lysis
16. Place the centrifuge rotor into a biological safety cabinet and carefully remove the bottles from the rotor being careful not to disturb the viral pellets, which may be invisible.
 

SECTION: Virus concentration and lysis
17. Pour off the supernatant and dispose of this as biohazardous waste, keeping the pellet in the bottom of the bottle.
SECTION: Initial sample clarification
7. Centrifuge the samples and controls at 10000 x g, 30 min at ambient temperature.

SECTION: Virus concentration and lysis
18. Add 2 mL of NucliSENS lysis buffer directly to the pellet.
 


SECTION: Virus concentration and lysis
19. Resuspend the pellet in lysis buffer thoroughly by repeat pipetting. Be careful not to introduce air into the lysis buffer at this stage to avoid foaming.
SECTION: Virus concentration and lysis
20. If RNA extraction is not to be carried out immediately, pipette the lysis buffer and pellet mixture into clean tubes and store at 3±2°C for up to 48 hours before proceeding to RNA extraction.
SECTION: RNA extraction
21. Transfer the lysis buffer and pellet mixture to a clean 24 well deep-well plate and place a 24-tip rack on the plate. Label this plate "Tips and lysate".
SECTION: RNA extraction
22. To the same number of wells as samples + controls in a 24 well deep-well plate, add 50 µL of well-mixed NucliSENS magnetic silica beads. Label this plate "Beads".
 


SECTION: RNA extraction
23. To the wells of two additional, clean 24 well deep-well plates, add 385 µL of NucliSENS wash buffer 1 to wells corresponding to those with magnetic silica beads prepared in step 23. Label these plates "Wash 1a and Wash 1b".

SECTION: RNA extraction
24. To the wells of two additional, clean 24 well deep-well plates, add 485 µL of NucliSENS wash buffer 2 to wells corresponding to those with magnetic silica beads prepared in step 23. Label these plates "Wash 2a and Wash 2b".

SECTION: RNA extraction
25. To the wells of one additional, clean 24 well deep-well plates, add 500 µL of NucliSENS wash buffer 3 to wells corresponding to those with magnetic silica beads prepared in step 23. Label this plates "Wash 3".

SECTION: RNA extraction
26. To the wells of one additional, clean 24 well deep-well plates, add 120 µL of NucliSENS wash buffer 3 to wells corresponding to those with magnetic silica beads prepared in step 23. Label this plate "Elution".
 


SECTION: RNA extraction
27. Load the WW_Nucisens_Kingfisher_24 protocol on the KingFisher™ Flex Purification System.

SECTION: RNA extraction
28. Load the wash buffer and sample/magnetic silica bead plates onto the KingFisher™ Flex Purification System and press Start.
SECTION: RNA extraction
29. Following the run, recover the elution plate and cover it with an adhesive cover.

SECTION: RNA extraction
30. If the RNA will not be used immediately for RT-qPCR following RNA purification, it should be stored at 3±2°C if it is intended to be used on the same day, <-15 °C for use within 1 week or ≤-70 °C for longer periods.

SECTION: RNA extraction
31. Decontaminate the KingFisher™ Flex Purification System to remove nucleases after each run using Thermo Scientific™ RNase AWAY™ Surface Decontaminant or a similar product.
SECTION: RT-qPCR
32. Make enough RT-qPCR master mix to test each sample (including total blank and phi6 positive controls), at least four ssRNA standard dilutions and TEX buffer NTC in at least duplicate for each assay (SARS-CoV-2 and phi6).
 

SECTION: RT-qPCR
33. Add the appropriate amount of RT-qPCR master mix to each well to be used in a PCR plate, leaving space for template RNA.
SECTION: RT-qPCR
34. Add sample RNA, control template RNA or NTC to the appropriate wells. For the N1 assay the volume used is 5 µL for phi6, used and 2 µL.

SECTION: RT-qPCR
35. Add ssRNA standard dilutions to the appropriate wells.
SECTION: RT-qPCR
36. Cover RT-qPCR plates and carry out RT-qPCR in a real-time PCR machine capable of supporting probe-based chemistry using the cycling conditions outlined in Table 4.

Table 4: Thermal cycling conditions for RT-qPCR 
 
 

SECTION: RT-qPCR
37. Note the time when the RT-qPCR run finishes.
SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
38. 
 
Open a vial of freeze-dried Pseudomonas culture from DSMZ and rehydrate in 0.5 mL TSB.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
39. Leave to rehydrate for 10 min and add to 8.5 mL of TSB.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
40. Incubate, shaking at 25±1°C 10 min (approximately 18 to 24 hours).

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
41. Check that the culture has become turbid 10 min, indicating bacterial growth.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
42. Add 1 mL of sterile glycerol and mix thoroughly by inverting several times.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
43. Split into 150 µL aliquots in screwcap cryovials and store at ≤ -70 °C for up to 1 year.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
44. New host stocks can be created by subculturing the host stock on TSA and using a single colony in place of the rehydrated Pseudomonas culture.
SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
45. Completely new host stocks should be prepared annually using a new vial of freeze-dried culture.
SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
46. 
 

Remove an aliquot of host stock from the freezer and allow it to thaw.
SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
47. Mix gently by flicking the side of the vial.
SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
48. Add 100 µL of host stock to 10 mL TSB in a sterile centrifuge tube and incubate, shaking at 25±1°C 10 min (approximately 18 to 24 hours).

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
49. Check that the culture has become turbid overnight, indicating bacterial growth.
 

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
50. Add 5 mL of TSB and 5 µL of 0.5 M calcium chloride solution to each of two centrifuge tubes.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
51. Add 0.5 mL of overnight host culture to each centrifuge tube, incubate, shaking at 25±1°C 10 min (approximately 18 to 24 hours).

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
52. Check that the culture has become turbid 10 min, indicating bacterial growth.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
53. Open and rehydrate a freeze-dried vial of phi6 and rehydrate in 500 µL Ringer’s ¼ Strength Solution.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
54. Add 100 µL of phi6 suspension to one of the host culture tubes, incubate both culture tubes, shaking at 25±1°C 10 min (approximately 18 to 24 hours).

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
55. The culture broth with added phi6 should look clearer than the control broth following incubation.
 

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
56. Centrifuge the phi6 culture at 4000 x g to 10000 x g for 15 min at 4±1°C and discard the control culture.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
57. Add 0.8 g of sterile glycerol to two 15 ml tubes, and then fill each to 6 mL with the supernatant and invert to mix.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
58. Split into 10 µL aliquots and store at <-70 °C for up to one year.

SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
59. New phi6 stocks should not be created by subculturing directly from broth cultures. Instead, the phi6 stock should be cultured by plaque assay according to Prussin et al. (2018) and individual plaques picked to be used as the starting phi6 material for step 55.
SECTION: Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
60. Completely new phi6 stocks should be created annually using a new vial of freeze-dried culture.