Apr 05, 2022
Version 2
RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500 V.2
- 1FDA
Protocol Citation: Jacquelina.Woods, rachel.rodriguez 2022. RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrdj4bgmk/v2Version created by Jessica L Jones
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 05, 2022
Last Modified: April 05, 2022
Protocol Integer ID: 60332
Keywords: GenomeTrakr, wastewater, SARS-CoV-2, N gene, crAssphage, murine norovirus, process control, extraction control, endogenous control, RT-qPCR, AB 7500 Fast
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Abstract
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes triplex and duplex assays for the RT-qPCR detection of the nucleocapsid region of the SARS-CoV-2 genome. These assays, along with the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay (RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater (protocols.io)), were developed for use on the AB 7500 platform using software version 2.0 or 2.3. All assays incorporate an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. These multiplexed detection assays were developed for the qualitative determination SARS-CoV-2 nucleocapsid gene extracted from wastewater. Valid sample results are contingent upon the detection of the MNV extraction control from the sample being tested.
Materials
Equipment and Supplies:
1. 7500 Fast Real-Time PCR System (ThermoFisher 4351106)
2. MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL (ThermoFisher 4346906), or MicroAmp Fast 8-Tube Strip, 0.1mL (ThermoFisher 4358293)
3. MicroAmp Optical Adhesive Film (ThermoFisher 4311971), or MicroAmp Optical 8-Cap Strips (ThermoFisher 4323032)
4. Platefuge (Fisher Scientific NC1823435), or Stripfuge (USA Scientific 2621-0016), or equivalent for either
5. 96-well cool block (USA Scientific 4051-0525, or equivalent)
6. Reagent cool block (USA Scientific 2312-2721, or equivalent) or ice bucket with ice
7. Adjustable calibrated micropipettes (0.2 – 1000 µl), two separate sets; one set dedicated for master mix setup and the other for template addition
8. Aerosol resistant pipet tips (0.2 – 1000 µl)
9. Personal microfuge (Labsource C90-044, or equivalent)
10. Hype-Wipe Disinfecting Towelettes (Fisher Scientific 14-412-56, or equivalent)
11. Vortex mixer (Labsource S16-200 or equivalent)
Reagents:
SARS-CoV-2 Genomic RNAATCCCatalog #1992D
Internal Control RNABioGXCatalog #750-0001((Contact sales@biogx.com for ordering)
One-Step RT-qPCR KitQiagenCatalog #210210 or 210212
50mM MgCl2Bio-rad LaboratoriesCatalog #17088872 or 25 mM MgCl2Thermo Fisher ScientificCatalog #AB0359
Nuclease-free waterLife TechnologiesCatalog #AM9937
Superase-In Life TechnologiesCatalog #AM2696
FAM reference dyeBio-rad LaboratoriesCatalog #1708780 , or equivalent, if running the N1-N2-IC triplex assay
ROX reference dyeFisher ScientificCatalog #12-223-012 , if running the N1-IC and N2-IC duplex assays
Tris (1 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9856
EDTA (0.5 M) pH 8.0 RNase-freeThermo Fisher ScientificCatalog #AM9261
Primers and probe sequences in following table. Custom Primers and ProbesIDT Technologies .
#based on accession nos. JF320650, MT006214.1, and NC_045512
aLuet al., 2020
bDepaola, Jones, Woods et. al. 2010, U.S. Patent Application 0060166232
cFluorophore for N1 and N2 should be FAM when running duplex assays, or JOE and Cy5, respectively, if running the triplex assay
*IB RQ- Iowa Black RQ
Before start
Always wear gloves during this procedure and never wear the same gloves when going between master mix and samples.
Always use aerosol resistant pipette tips for PCR.
Note
Detection of SARS-CoV-2 can be conducted as a triplex assay (N1-N2-IAC), or as two duplex assays (N1-IAC and N2-IAC).
Safety information
Assembly of master mix should be done in a designated Master Mix PCR hood or BSC that has been decontaminated with 10% Bleach solution or HypeWipes followed by 70% Ethanol, or similar product and UV irradiated for 20 minutes prior to use. RNA sample template should be added in a separate designated area, physically separated from the Master Mix hood/area. Equipment should not be shared between the two areas.
Master Mix Preparation
Master Mix Preparation
5s
5s
Prepare Master Mix for all sample and control reactions based on the volumes per 25 µl reaction in this table. Composition of mixes are listed here: Reagent Mixes for RT-qPCR Detection of SARS-CoV-2 from Wastewater (protocols.io) and should be prepared in advance and stored appropriately. Alternatively, Master Mixes can be prepared from individual components as described here: 
Master Mix Tables for SARS-CoV-2 Assays .pdf .
Master Mix Tables for SARS-CoV-2 Assays .pdf . A Use a 1:1000 dilution (made in Primer TE) of FAM reference dye in the N1-N2-IC triplex assay.
B Use a 1:10 dilution (made in Primer TE) of ROX reference dye in the N1-IC and N2-IC duplex assays.
*Amount varies with concentration of IC RNA. The amount of IC RNA template needs to be adjusted based on the prepared stock concentration to report a Cycle threshold (Ct) of 20-25 when no inhibition is present in the reaction (i.e., the negative control reaction).
Safety information
Do not add IC or sample RNA at this step!
Thaw Master Mix reagents in bench top cool block (chilled at 2-8 °C ) or On ice in master mix preparation hood.
Safety information
Keep Enzyme chilled continually; these enzymes are in glycerol and do not need to be thawed.
Vortex reagent tubes for 00:00:03 +/- 1 sec at setting medium high to high (if vortex has settings).
3s
Briefly centrifuge all reagents 00:00:05 +/- 2 sec in a personal microcentrifuge to bring liquid to the bottom of tube.
5s
Return all reagents to bench top cool block (chilled at 2-8 °C ) or On ice .
Proceed to hood/area or room where the template is added and thaw IC RNA and sample RNA in this hood/area.
Safety information
RNA templates should be added to reaction tubes in a designated area separate from location where master mixes are prepared. A negative (water) and positive PCR control should be added to each reaction set-up.
Briefly centrifuge IC RNA 00:00:05 +/- 2 sec in a personal microcentrifuge to bring liquid to the bottom of tube.
5s
Add appropriate volume of IC RNA (0.2 µL per reaction) to Master Mix from Step 1.4 in cold block/on ice.
Vortex briefly and centrifuge 00:00:05 +/- 2 sec in a personal microcentrifuge.
5s
Reaction Set-Up
Reaction Set-Up
5s
5s
Add 22 µL of Master Mix to each designated reaction tube or sample wells.
Briefly centrifuge sample RNA 00:00:05 +/- 2 sec in a personal microcentrifuge to bring liquid to the bottom of tube.
5s
Add 3 µL of sample RNA template to each of three reaction tubes or wells.
Ensure each plate or run has appropriate controls (positive and negative controls) included.
Note
Positive control prepared as described here: Positive Control Material for RT-qPCR of SARS-CoV-2 and Process Controls (protocols.io).
Seal sample plate or strip tubes. Then, briefly spin 00:00:05 +/- 2 sec .
5s
Start run on Applied Biosystems 7500 Fast instrument.
Note
Assay parameters were optimized using the AB 7500 software versions 2.0-2.3. If other instruments or software versions are used, additional optimization may be needed.
Use the following settings for the Experiment Properties:
"7500Fast (96 wells)"
"Quantitation Standard Curve"
"TaqMan Reagents"
"Standard (~2 hours to complete run)"
Identify the appropriate target reporters and leave all quenchers as "NFQ-MGB".
Safety information
Example in image is for the N1-N2-IAC triplex assay (N1-Joe, N2-Cy5, IC-Texas Red). If using the duplex assays, adjust appropriately (N1-FAM, N2-FAM, IC-Cy5).
Select appropriate passive reference dye (FAM for the triplex assay).
Safety information
Example in image is for the N1-N2-IAC triplex assay. If using the duplex assays, adjust appropriately (ROX).
Assign targets and samples.
Use the following settings for Run Method:
25 µL reaction volume
Holding stage 1: 50 °C for 00:50:00
Holding stage 2: 95 °C for 00:15:00
Cycling stage: 45 cycles of 95 °C for 00:00:10 , 56 °C for 00:00:25 , 62 °C for 00:01:00
Enable data collection on Step 3 of Cycling stage
Note
These are the cycling conditions for the N1-N2-IAC triplex as well as the N1-IAC and N2-IAC duplex assays.
1h 6m 35s
Data Analysis
Data Analysis
Adjust analysis settings to appropriate thresholds. For the triplex assay, N1 and N2 should be set at 0.01 and the IC set at 0.1. Baseline start cycle should be set at 3 and baseline end cycle should be set at 10.
Safety information
Example in image is for the N1-N2-IAC triplex assay. If using the duplex assays, adjust appropriately (all thresholds set at 0.01).
Verify positive and negative calls for each reaction using either linear or log amplification plots.
RT-qPCR run is invalid if any of the following are observed:
1. Negative RT-qPCR control is positive (Ct value indicated) for any of the expected SARS-CoV-2 targets;
2. Positive RT-qPCR control is negative (undetermined) for expected SARS-CoV-2 targets; or
3. IC is negative (undetermined) in the negative RT-qPCR control; or
Note
Run is invalid and the RT-qPCR assay must be repeated.
Sample is negative if:
1. Negative and positive RT-qPCR control reactions give appropriate results;
2. Sample reaction is negative (undetermined) for expected SARS-CoV-2 target/s; and
3. Internal amplification control (IC) is positive.
Safety information
Valid SARS-CoV-2 results require the successful detection of MNV extraction control from the sample as described in *link protocol*. If MNV is negative for a sample, that sample is invalid and should not be reported as negative.
Sample is positive if:
1. Negative and positive RT-qPCR control reactions give appropriate results; and
2. Sample reaction is positive (Ct value indicated) for expected SARS-CoV-2 target/s.
Note
If at least one of the triplicate sample reactions are positive, the sample should be reported as positive.