RT-qPCR for detection of SARS-CoV-2 in wastewater
Livia C T Scorza, David Findlay, Julie Bolland, Brindusa Cerghizan, Kirsty Campbell, David Thomson, Alexander Corbishley, David Gally, Stephen Fitzgerald, Alison Tidswell, Sean McAteer
Wastewater-based epidemiology
Infectious diseases
Public health
Covid-19
SARS-CoV-2
RNA viruses
RT-PCR
Abstract
As part of the global response to the 2019 novel Coronavirus (SARS-CoV-2) pandemic, it was determined that SARS-CoV-2 RNA was detectable in the faeces of both symptomatic and asymptomatic patients (1). Further analysis demonstrated that a wastewater epidemiological (WWE) approach, similar to that used to track other viruses (i.e. Poliovirus), could be employed to monitor the spread of SARS-CoV-2. The presence of, or changes in concentration of viral RNA within the wastewater network can assist in monitoring the emergence of further viral peaks (2). Thus, monitoring the spread of Covid-19 using the WWE approach has been extensively explored in several countries (3). Viral RNA is extracted from a known volume of sewage treatment works influent and amplified by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) RT-qPCR is a process whereby RNA is first converted to complementary DNA (cDNA) after which it can undergo amplification and quantification via PCR. The amplification of a non-target sequence, in this case the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), can also be carried out to act as an internal control.This procedure, developed by Scottish Environment Protection Agency (SEPA) based on work in the Corbishley lab, Roslin Institute of the University of Edinburgh, outlines the method for amplification and quantification of the RNA extracted from crude sewage samples, but could equally apply to other RNA extracted from other types of environmental sample.For viral RNA extraction from wastewater, please search for "RNA extraction from wastewater for detection of SARS-CoV-2" by the same authors of this protocol on protocols.io References:1. Jones, D. L., Baluja, M. Q., Graham, D. W., Corbishley, A., McDonald, J. E., Malham, S. K., Hillary, L. S., Connor, T. R., Gaze , W. H., Moura , I. B., Wilcox, M. H., & Farkas , K. (2020).Shedding of SARS-CoV-2 in feces and urine and its potential role in person-to-person transmission and the environment-based spread of COVID-19. Science of the Total Environment .https://doi.org/10.1016/j.scitotenv.2020.1413644.2. Fitzgerald, S., Rossi, G., Low, A., McAteer, S., O’Keefe, B., Findlay, D., Cameron, G. J., Pollard, P., Singleton, P. T. R., Ponton, G., Singer, A. C., Farkas, K., Jones, D., Graham, D. W., Quintela-Baluja, M., Tait-Burkard, C., Gally, D., Kao, R., & Corbishley, A.(2021).Site specific relationships between COVID-19 cases and SARS-CoV-2 viral load in wastewater treatment plant influent. Environmental Science and Technology . https://doi.org/10.1021/acs.est.1c050293. Wastewater SARS Public Health Environmental Response (W-SPHERE). https://sphere.waterpathogens.org/about
Before start
Samples should be processed as soon as possible after extraction. If previously frozen they must be analysed immediately after they are defrosted (on ice).
Steps
Sample and equipment setup
At start of day, clean all surfaces with RNase away or 10% v/v bleach solution.
Turn on UV lamps in the Master Mix preparation cabinet and Template addition cabinet for 20 minutes.
Prior to analysis; ensure there are sufficient cooler blocks and/or ice baths for each workstation.
Complete RT-qPCR Plate Worksheet and calculate the number of reactions (N) and volume of Master Mix required. See Appendix 1 for details. Appendix_1_SARS-CoV-2_RT-qPCR_plate_template.xlsx
Defrost sufficient RT-qPCR reagents and refrigerate until ready to use.
N.B. DO NOT REMOVE RT ENZYME FROM -20°C FREEZER UNTIL READY TO USE. MAINTAIN ON ICE AT ALL TIMES AND RETURN IMMEDIATELY AFTERWARDS.
Defrost positive control and any required samples and maintain on ice.
Standard curve preparation
For the standard curve preparation, use a 1:10 dilution series covering 5 dilution points (50000 to 5 copies of SARS-CoV2 ssRNA).
In positive template cabinet label 5 tubes and add 225 µl of RNase-free water to each and place on ice.
Transfer 25 µl from stock solution and add to labelled (5e+4) tube.
Mix and spin twice to ensure thorough homogenisation
Repeat step 9 and 10 until dilution series is complete. Store on ice until needed.
Go to "Standard curve analytical Control" steps for data interpretation (32 - 33)
Master Mix Preparation
DEFROST AND MAINTAIN REAGENTS ON ICE
| A | B | C |
|---|---|---|
| Reagent | Volume (µl) per reaction (N) | Per sample (duplicate x 2.5) |
| RT-qPCR reaction mix | 10 | 25 |
| RT enzyme | 1 | 2.5 |
| Primer/Probe mix | 1.5 | 3.75 |
| BSA | 2 | 5 |
| Nuclease-free water | 0.5 | 1.25 |
| Total of master mix (without template) | 15 | 37.5 |
| Template RNA | 5 | n/a |
| Total reaction volume | 20 | 50 |
Table 1 Master Mix
The list containing primers and probes sequences and concentration can be found in the attached file below
In the Master Mix cabinet, working on ice, combine the master mix reagents (excluding template). Invert x5 to mix.
Dispense 15 µl master mix to each well required of a 96 well PCR plate (on ice).
Add 5 µl of nuclease-free water to "no template control" (NTC) wells and cap.
Cover plate and transfer to sample addition cabinet
Plate setup
Add 5 µl of sample to appropriate wells and cap.
Cover and transfer to Template addition cabinet.
Add 5 µl of each positive control dilution series to appropriate wells and cap.
Spin plate down (1000 RCF for 1 minute) and transfer the plate to the qPCR instrument. Lower and lock the upper tray in place and close the door
RT-qPCR using AriaMx Real-Time PCR system
Select “New Experiment” and “Quantitative PCR (Fluorescence Probe)”.
In “Plate setup” select all wells and assign as per Plate Worksheet and choose relevant filters
N1 – FAM and ROX.
E gene (optional) – HEX and ROX
PRRS – Cy5 and ROX
In Thermal Profile tab set the following parameters:
| A | B | C |
|---|---|---|
| Temperature | Time | Cycles |
| 55 ºC | 10 min | 1 |
| 95 ºC | 1 min | 1 |
| 95 ºC | 10 seconds | 40 |
| 55 ºC | 30 seconds |
RT-qPCR parameters
Save file with desired name
Select "Run experiment"
After Run Save file and copy to PC for detailed plate setup.
Open file using Aria software
Highlight all wells and assign ROX as reference dye.
Highlight sample wells and assign sample identifier (this can be imported from qPCR worksheet XL).
Highlight “Standard” wells and assign appropriate concentration values
Standard curve analytical quality control
Before results can be determined highlight all wells and select the following columns: Well, Well Name, Well Type, Ct (dRn) or Cq (∆Rn), Quantity (copies), RSq (dRn) or R² (∆Rn), Slope (dRn) and Efficiency (%).
Check the baseline threshold values (~0.03 FAM and ~0,1 Cy5).
Ensure that NTC wells are negative (No Cq).
Ensure that Standard Curve meets the following criteria:
R-Sq is >0.9,
Slope (y) is between -3.1 and -3.92 and
Efficiency is between 80 and 110%.
If any of these parameters are exceeded then analysis should be repeated.
Interpretation of raw data
Once QC data has been checked raw data can be exported to XL where the number of gene copy equivalent per litre can be calculated.
Results should be reported as follows:
1.Replicate mean >LoQ = Quantifiable Positive Result (ie 12345 gc/L)
2.Replicate mean >LoD but <LoQ = “Positive (DNQ)”
3.Replicate mean <LoD = “Weak Positive”
4.If both replicates produce No Cq result = “Negative”
LoQ= limit of quantification
LoD= limit of detection
Check "Guidelines & Warnings" section for more details on the LoQ and LoD values
