Quantification of SARS-CoV-2 in wastewater
James Lowther, David Ian Walker, Nick Evens, Jonathan Warren, Jonathan Porter, Kata Farkas, Davey Jones
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Abstract
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.
Attachments
Steps
Preparation of daily phi6 process control working suspensions
On each day of testing, a new batch of phi6 process control working suspension should be made.
Remove an aliquot of phi6 stock from the freezer (prepared according to Appendix 1), thaw and mix thoroughly by vortexing.
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.
Initial sample clarification
Mix each sample by inverting the sample bottle 10 times within a biological safety cabinet.
Measure approximately 200mL of sample into a suitable centrifuge bottle.
Prepare at least two control centrifuge bottles with approximately 200mL of spring water on each day of testing.
Centrifuge the samples and controls at 10000x g at ambient temperature.
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.
Virus concentration and lysis
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.
To each 150mL 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.
Pour the sample/phi6 mixtures in to centrifuge bottles containing 60±1 g of ammonium sulphate.
Dissolve the ammonium sulphate by inverting the bottles several times, or by shaking on an orbital shaker at approximately 200rpm,0h 0m 0s until all of the ammonium sulphate has dissolved.
Incubate the samples at 3±2°C for at least 60 to 180 minutes.
Mark the outer sides of the bottle where the pellets will form during centrifugation.
Centrifuge the chilled samples for 0h 30m 0s at 10000x g,0h 0m 0s at 4±1°C with no braking.
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.
Pour off the supernatant and dispose of this as biohazardous waste, keeping the pellet in the bottom of the bottle.
Add 2mL of NucliSENS lysis buffer directly to the pellet.
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.
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.
RNA extraction
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".
To the same number of wells as samples + controls in a 24 well deep-well plate, add 50µL of well-mixed NucliSENS magnetic silica beads. Label this plate "Beads".
To the wells of two additional, clean 24 well deep-well plates, add 385µL 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".
To the wells of two additional, clean 24 well deep-well plates, add 485µL 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".
To the wells of one additional, clean 24 well deep-well plates, add 500µL of NucliSENS wash buffer 3 to wells corresponding to those with magnetic silica beads prepared in step 23. Label this plates "Wash 3".
To the wells of one additional, clean 24 well deep-well plates, add 120µL of NucliSENS wash buffer 3 to wells corresponding to those with magnetic silica beads prepared in step 23. Label this plate "Elution".
Load the WW_Nucisens_Kingfisher_24 protocol on the KingFisher™ Flex Purification System.
Load the wash buffer and sample/magnetic silica bead plates onto the KingFisher™ Flex Purification System and press Start.
Following the run, recover the elution plate and cover it with an adhesive cover.
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.
Decontaminate the KingFisher™ Flex Purification System to remove nucleases after each run using Thermo Scientific™ RNase AWAY™ Surface Decontaminant or a similar product.
RT-qPCR
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).
Add the appropriate amount of RT-qPCR master mix to each well to be used in a PCR plate, leaving space for template RNA.
Add sample RNA, control template RNA or NTC to the appropriate wells. For the N1 assay the volume used is 5µL for phi6, used and 2µL.
Add ssRNA standard dilutions to the appropriate wells.
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
| A | B | C |
|---|---|---|
| Temperature | Time | Cycles |
| 52°C | 20 minutes | 1 |
| 96°C | 10 minutes | 1 |
| 94°C | 15 seconds | 45 |
| 60°C | 1 minutes |
Note the time when the RT-qPCR run finishes.
Appendix 1 Preparation of phi6 process control material: Preparation of Pseudomonas host stock
0.5mL TSB. Leave to rehydrate for 0h 10m 0s and add to 8.5mL of TSB.
Incubate, shaking at 25±1°C 0h 10m 0s (approximately 18 to 24 hours).
Check that the culture has become turbid 0h 10m 0s, indicating bacterial growth.
Add 1mL of sterile glycerol and mix thoroughly by inverting several times.
Split into 150µL aliquots in screwcap cryovials and store at ≤ -70°C for up to 1 year.
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.
Completely new host stocks should be prepared annually using a new vial of freeze-dried culture.
Appendix 1 Preparation of phi6 process control material: Preparation of phi6 stock
Remove an aliquot of host stock from the freezer and allow it to thaw.
Mix gently by flicking the side of the vial.
Add 100µL of host stock to 10mL TSB in a sterile centrifuge tube and incubate, shaking at 25±1°C 0h 10m 0s (approximately 18 to 24 hours).
Check that the culture has become turbid overnight, indicating bacterial growth.
Add 5mL of TSB and 5µL of 0.5 M calcium chloride solution to each of two centrifuge tubes.
Add 0.5mL of overnight host culture to each centrifuge tube, incubate, shaking at 25±1°C 0h 10m 0s (approximately 18 to 24 hours).
Check that the culture has become turbid 0h 10m 0s, indicating bacterial growth.
Open and rehydrate a freeze-dried vial of phi6 and rehydrate in 500µL Ringer’s ¼ Strength Solution.
Add 100µL of phi6 suspension to one of the host culture tubes, incubate both culture tubes, shaking at 25±1°C 0h 10m 0s (approximately 18 to 24 hours).
The culture broth with added phi6 should look clearer than the control broth following incubation.
Centrifuge the phi6 culture at 4000x g,0h 0m 0s to 10000x g,0h 0m 0s for 0h 15m 0s at 4±1°C and discard the control culture.
Add 0.8g of sterile glycerol to two 15 ml tubes, and then fill each to 6mL with the supernatant and invert to mix.
Split into 10µL aliquots and store at <-70°C for up to one year.
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.
Completely new phi6 stocks should be created annually using a new vial of freeze-dried culture.
