Nov 16, 2023
Version 1
Fecal sample pooling for SARS-CoV-2 real-time RT-PCR screening V.1
- Leyi Wang1,
- Antonio Leonardi-Cattolica1,
- Sandipty Kayastha1,
- Megan Miller2
- 1University of Illinois at Urbana Champaign;
- 2U.S. Food and Drug Administration
Protocol Citation: Leyi Wang, Antonio Leonardi-Cattolica, Sandipty Kayastha, Megan Miller 2023. Fecal sample pooling for SARS-CoV-2 real-time RT-PCR screening. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3oobl3p/v1
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: June 06, 2023
Last Modified: November 16, 2023
Protocol Integer ID: 82960
Keywords: Feces, sample pooling, SARS-CoV-2
Funders Acknowledgements:
Vet-LIRN
Grant ID: 1 U18 FD007727-01
Disclaimer
Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
Abstract
This document includes the information and procedures to do sample pooling for fecal samples and real-time RT-PCR for SARS- CoV-2 using CDC based primers and probes.
Validation data for 5 sample pools (in-house and by an independent laboratory via collaborative study such as Blinded Method Test) are available upon request.
Materials
A. Kits and Reagents
- CDC-based SARS-CoV-2 N1 primers and probe
2019-nCoV_N1 Cimbined Primer/Probe Mix at 22.5 nmol
- MagMAX™ Pathogen RNA/DNA Kit, Catalog number: 4462359.
- ThermoFisher Scientific. AgPath-ID™ One‑Step RT‑PCR Reagents, Catalog number: AM1005.
B. Equipment
- KingFisher 96 magnetic particle processor, or equivalent (Thermo Electron Corp. Cat#5400050)
- Benchtop centrifuge-Eppendorf (Model# 5415 D) or equivalent
- BioRad CFX96, BioRad Opus real-time PCR machine or equivalent
- Vortex Mixer or equivalent
- Mini Plate Spinner, equivalent, or larger centrifuge capable of centrifuging 96-well plates
C. Supplies
- 1000 μl, 200 μl, and 20 μl pipets
- 1000 μl, 200 μl and 20 μl aerosol pipet tips
- Serological pipettor
- 50 ml reservoirs
- Sterile 1.5 ml microcentrifuge tubes
- Sterile PCR reaction tubes, strip tubes, or 96-well plates
- Optically clear adhesive seals for plates
- Gloves
- 10 ml, 5 ml serological pipets
Safety warnings
SARS-CoV-2 is a Biosafety Level 3 agent and samples should be handled as approved by each insitution. Proper PPE including double gloves, disposable or autoclavable gown, N95 with face shield or CAPR unit is required and must be used when working in the BSL-3. Gloves and lab coats are required for extraction and PCR setup. Gloves must be worn at any time when touching extraction or PCR plates or reagents.
Prepare fecal samples
Prepare fecal samples
Add 1 mL phosphate buffered saline (PBS, pH 7.4) to an Eppendorf tube.
Use cotton tipped wooden swabs to wipe a fecal sample in four places.
Note
Consistence of fecal samples can vary. For drier samples cotton tipped can be dipped into PBS prior to wiping the fecal samples.
Place the cotton swab with fecal material into the PBS in the Eppendorf tube.
Move the swab up and down within the PBS three time to remove as much feces as possible.
While removing the swab, let swab contact the top inside of Eppendorf tube with slight pressure to avoid losing solution.
Note
Some PBS will remain on the swab after removal.
Add additional amount of PBS to make1 mL fecal suspension solution for each sample.
Vortex the tube for 00:00:15 and 8000 rpm centrifuge00:02:00 .
2m 15s
Sample can be stored at -20/-80C
Note
-80C is recommended to prevent viral degradation
Sample pooling
Sample pooling
Based on the manual procedure of MagMAX™ Pathogen RNA/DNA Kit, 200 µl amount of fecal suspension supernatant is used for extraction of nucleic acid.
For a 5-sample pooling: 40 µL of supernatant from each sample will be mixed in a new Eppendorf tube, for a total of 200 µL
Note
Critical point: Make sure all material is transferred into micro plate
Note
If samples were stored after step 5 make sure to thaw on ice and repeat step 5
For a 10-sample pooling,20 µL of supernatant from each sample will be mixed in a new Eppendorf tube, for a total of200 µL .
Note
Critical point: Make sure all material is transferred into micro plate
RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
Prepare lysis/binding solution and mix well by vortexing
Note
Amount needed for each sample are given below. Make a master mix by combining all elements into one solution for total samples being processed.
Lysis/Binding Solution Concentrate: 200 µL
Carrier RNA (μg/μL): 2 µL
100% Isopropanol: 200 µL
Prepare the Bead Mix and mix well by vortexing
Note
Amount needed for each sample are given below. Make a master mix by combining all elements into one solution for total samples being processed.
Nucleic Acid Binding Beads: 10 µL
Lysis ENHANCER: 10 µL
Set up plates.
Lable a MME-96 Deep Well Plate "First Wash 1" and add 300 µL into each well with Wash Soultion 1
Lable a MME-96 Deep Well Plate "Second Wash 1" and add 300 µL into each well with Wash Soultion 1
Lable a MME-96 Deep Well Plate "First Wash 2" and add 450 µL into each well with Wash Soultion 2
Lable a MME-96 Deep Well Plate "Second Wash 2" and add 450 µL into each well with Wash Soultion 2
Lable a MME-96 Deep Well Plate "Elution". Add60 µL Elution buffer to each well.
Put Tip comb plate to a MME-96 Standard Plate.
Set up sample plate in a MME-96 Deep Well Plate.
Add 20 µL of prepared Bead Mix to each well.
add 200 µL of fecal suspension supernatant mixtures to each well.
add 400 µL lysis/binding solution to each well.
Vortex sample plate on shaker for 00:05:00
5m
Load all plates to KingFisher Flex for extraction of nucleic acid using the MagMAX™ Pathogen RNA/DNA Kit procedure.
Select the program: MagMax_Pathogen_Hight_Volume 96DW on the instrument (4462359_DW_50)
Once extraction is complete, take the elution plate out.
Store the purified nucleic acid on ice for immediate use, at –20°C for
up to 1 month, or at –80°C for long‑term storage.
Real-time RT-PCR
Real-time RT-PCR
AgPath-ID™ One-Step RT-PCR Reagents is used in the real-time RT-PCR along with the CDC-based SARS-CoV-2 N1 primer assay
Note
Amount needed for each sample are given below. Make a "master mix' by combining all elements into one solution for total samples being processed.
2X RT‑PCR Buffer: 12.5 µL
25X RT‑PCR Enzyme Mix: 1 µL
CDC-based SARS-CoV-2 N1 primers and probe (FAM dye) are used: 2 µL
The VetMAX™ Xeno™ Internal Positive Control (IPC) - LIZ™ Assay (Cy5): 1 µL
VetMAX™ Xeno™ Internal Positive Control DNA: 1 µL
DNase/RNase-free distilled water: 2.5 µL
Add 20 µL of Master Mix to each well/tube used for RT-PCR
Add 5 µL RNA templates pooled samples, Negative template control (NTC) such as, DNase/RNase-free distilled Water, and positive control.
Program real time thermocycler
48oC for 10 minutes.
95 oC for 10 minutes.
40 cycles of
95oC for 15 seconds.
60oC for 45 seconds.
8. Data analysis.
8. Data analysis.
Check Ct value of Xeno spiked control for each sample to see if any inhibition occurs
Protocol references
Applied Biosystem. ThermoFisher Scientific. MagMAX™ Pathogen RNA/DNA Kit, Catalog number: 4462359. User Guide. Revision C.
Applied Biosystem. ThermoFisher Scientific. AgPath-ID™ One‑Step RT‑PCR Reagents, Core reagents for one-step qRT-PCR detection of pathogens. Catalog number: AM1005. USER GUIDE. Revision J
CDC 2019-Novel Coronavirus (2019 nCoV) Real-Time RT-PCR Diagnostic Panel . Catalog # 2019-nCoVEUA-01 1000 reactions. Center for Diease Control and Prevention