Fecal sample pooling for SARS-CoV-2 real-time RT-PCR screening
Leyi Wang, Antonio Leonardi-Cattolica, Sandipty Kayastha, Megan Miller
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.
Steps
Prepare fecal samples
Add 1mLphosphate buffered saline (PBS, pH 7.4) to an Eppendorf tube.
Use cotton tipped wooden swabs to wipe a fecal sample in four places.
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.
Add additional amount of PBS to make1mLfecal suspension solution for each sample.
Vortex the tube for 0h 0m 15s and 8000 rpm centrifuge0h 2m 0s.
Sample can be stored at -20/-80C
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: 40µLof supernatant from each sample will be mixed in a new Eppendorf tube, for a total of 200µL
For a 10-sample pooling,20µL of supernatant from each sample will be mixed in a new Eppendorf tube, for a total of200µL.
RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
Prepare lysis/binding solution and mix well by vortexing
Lysis/Binding Solution Concentrate: 200µL
Carrier RNA (μg/μL): 2µL
100% Isopropanol: 200µL
Prepare the Bead Mix and mix well by vortexing
Nucleic Acid Binding Beads: 10µL
Lysis ENHANCER: 10µL
Set up plates.
Lable a MME-96 Deep Well Plate "First Wash 1" and add 300µL into each well with Wash Soultion 1
Lable a MME-96 Deep Well Plate "Second Wash 1" and add 300µL into each well with Wash Soultion 1
Lable a MME-96 Deep Well Plate "First Wash 2" and add 450µL into each well with Wash Soultion 2
Lable a MME-96 Deep Well Plate "Second Wash 2" and add 450µL into each well with Wash Soultion 2
Lable a MME-96 Deep Well Plate "Elution". Add60µL 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 20µL of prepared Bead Mix to each well.
add 200µL of fecal suspension supernatant mixtures to each well.
add 400µL lysis/binding solution to each well.
Vortex sample plate on shaker for 0h 5m 0s
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
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
2X RT‑PCR Buffer: 12.5µL
25X RT‑PCR Enzyme Mix: 1µL
CDC-based SARS-CoV-2 N1 primers and probe (FAM dye) are used: 2µL
The VetMAX™ Xeno™ Internal Positive Control (IPC) - LIZ™ Assay (Cy5): 1µL
VetMAX™ Xeno™ Internal Positive Control DNA: 1µL
DNase/RNase-free distilled water: 2.5µL
Add 20µLof Master Mix to each well/tube used for RT-PCR
Add 5µLRNA 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.
95oC for 10 minutes.
40 cycles of
95oC for 15 seconds.
60oC for 45 seconds.
8. Data analysis.
Check Ct value of Xeno spiked control for each sample to see if any inhibition occurs
