Apr 05, 2022
Version 2
RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater using AB 7500 V.2
- 1FDA
Protocol Citation: Jacquelina.Woods, rachel.rodriguez 2022. RT-qPCR Detection of Process Controls (Murine noroviurs and crAssphage) from Wastewater using AB 7500. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg36j9pg25/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: 60337
Keywords: GenomeTrakr, wastewater, SARS-CoV-2, 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 the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay developed for use on the AB 7500 platform using software version 2.0 or 2.3. The assay incorporates an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. This multiplexed detection assay was developed for the determination crAssphage extracted from wastewater, as an endogenous control, and MNV as an extraction control. The assay is designed to be used in conjunction with the SARS-CoV-2 RT-qPCR detection assay. Valid sample results for SARS-CoV-2 detection 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
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:
Internal Control RNABioGXCatalog #750-0001((Contact sales@biogx.com for ordering)
Murine NorovirusATCCCatalog #VR 1937
One-Step RT-qPCR KitQiagenCatalog #210210 or 210212
Nuclease-free waterLife TechnologiesCatalog #AM9937
Superase-In Life TechnologiesCatalog #AM2696
FAM reference dyeBio-rad LaboratoriesCatalog #1708780 , or equivalent
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
aHewitt, et al., 2009
bModifiedfrom Stachler, et al., 2017
cDepaola, Jones, Woods et. al. 2010, U.S. Patent Application 0060166232
*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.
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
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 Process Controls from Wastewater Samples (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 Table for MNV-crAss Assay.pdf .
Master Mix Table for MNV-crAss Assay.pdf .A Use a 1:1000 dilution (made in Primer TE) of FAM reference dye in the N1-N2-IC triplex assay.
*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
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.
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 .
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 (MNV-Cy5, crAss-JOE, IC-TexasRed) and leave all quenchers as "NFQ-MGB".
Select appropriate passive reference dye (FAM for the triplex assay).
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 00:00:15 , 55 °C for 00:00:20 , 62 °C for 00:01:00
Enable data collection on Step 3 of Cycling stage
1h 6m 35s
Data Analysis
Data Analysis
Adjust analysis settings to appropriate thresholds. All thresholds should be set at 0.01. Baseline start cycle should be set at 3 and baseline end cycle should be set at 10.
Verify positive and negative calls for each reaction using either linear or log amplification plots.
Sample is invalid if any of the following are observed:
1. Negative RT-qPCR control is positive (Ct value indicated) for any of the expected targets (MNV or crAssphage);
2. Positive RT-qPCR control is negative (undetermined) for expected target/s (MNV or crAssphage);
3. Sample is negative (undetermined) for MNV; or
4. IC is negative (undetermined) in the sample, or the average of the IC Ct values from the sample replicates are greater than 4 Cts than the IC Ct of the negative RT-qPCR control.
Note
Sample is invalid and RT-qPCR should be repeated. If still invalid, an additional concentrate from Virus Concentration from Wastewater Using PEG Precipitation and Ultracentrifugation (protocols.io) should be extracted and tested.
Sample is valid if all of the following are observed:
1. Negative RT-qPCR control is negative (undetermined) for expected target/s (i.e., MNV and crAssphage);
2. Positive RT-qPCR control is positive (Ct value indicated) for expected target/s (i.e., MNV and crAssphage); and
3. Internal amplification control (IC) is positive in all sample reactions with the average IC Ct values for the sample is less than 4 Cts greater than the IC Ct of the negative RT-qPCR control.
Note
Sample is valid, proceed to determination of SARS-CoV-2 results as described RT-qPCR Detection of SARS-CoV-2 from Wastewater Using the AB 7500 (protocols.io).