TITLE: Fecal sample pooling for SARS-CoV-2 real-time RT-PCR screening
AUTHORS: Leyi Wang, Antonio Leonardi-Cattolica, Sandipty Kayastha, megan.miller

[DESCRIPTION]

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]
SECTION: Prepare fecal samples
1. Add 1 mLphosphate buffered saline (PBS, pH 7.4) to an Eppendorf tube.

SECTION: Prepare fecal samples
2. Use cotton tipped wooden swabs to wipe a fecal sample in four places.

 

SECTION: Prepare fecal samples
3. Place the cotton swab with fecal material into the PBS in the Eppendorf tube.
SECTION: Prepare fecal samples
3.1. Move the swab up and down within the PBS three time to remove as much feces as possible.
SECTION: Prepare fecal samples
3.2. While removing the swab, let swab contact the top inside of Eppendorf tube with slight pressure to avoid losing solution.
 

SECTION: Prepare fecal samples
4. Add additional amount of PBS to make1 mLfecal suspension solution for each sample.

SECTION: Prepare fecal samples
5. Vortex the tube for 15 s and 8000 rpm centrifuge2 min.

SECTION: Prepare fecal samples
6. Sample can be stored at -20/-80C
 

SECTION: Sample pooling
7. 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.
SECTION: Sample pooling
8. For a 5-sample pooling: 40 µLof supernatant from each sample will be mixed in a new Eppendorf tube, for a total of 200 µL
 

 



SECTION: Sample pooling
9. 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.
 



SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
10. Prepare lysis/binding solution and mix well by vortexing

 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
10.1. Lysis/Binding Solution Concentrate: 200 µL 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
10.2. Carrier RNA (μg/μL): 2 µL

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
10.3. 100% Isopropanol: 200 µL

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
11. Prepare the Bead Mix and mix well by vortexing

 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
11.1. Nucleic Acid Binding Beads: 10 µL

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
11.2. Lysis ENHANCER: 10 µL

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
12. Set up plates. 
SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
12.1. Lable a MME-96 Deep Well Plate "First Wash 1" and add 300 µL into each well with Wash Soultion 1 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
12.2. Lable a MME-96 Deep Well Plate "Second Wash 1" and add 300 µL into each well with Wash Soultion 1 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
12.3. Lable a MME-96 Deep Well Plate "First Wash 2" and add 450 µL into each well with Wash Soultion 2

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
12.4. Lable a MME-96 Deep Well Plate "Second Wash 2" and add 450 µL into each well with Wash Soultion 2

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
12.5. Lable a MME-96 Deep Well Plate "Elution". Add60 µL Elution buffer to each well.

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
12.6. Put Tip comb plate to a MME-96 Standard Plate.
SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
13. Set up sample plate in a MME-96 Deep Well Plate.
SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
13.1. Add 20 µL of prepared Bead Mix to each well. 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
13.2. add 200 µL of fecal suspension supernatant mixtures to each well. 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
13.3. add 400 µL lysis/binding solution to each well. 

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
13.4. Vortex sample plate on shaker for 5 min

SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
14. Load all plates to KingFisher Flex for extraction of nucleic acid using the MagMAX™ Pathogen RNA/DNA Kit procedure.
SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
15. Once extraction is complete, take the elution plate out. 
SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
14.1. Select the program: MagMax_Pathogen_Hight_Volume 96DW on the instrument (4462359_DW_50)
SECTION: RNA extraction using MagMAX™ Pathogen RNA/DNA Kit
15.1. 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.
SECTION: Real-time RT-PCR
16. 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 

 

SECTION: Real-time RT-PCR
16.1. 2X RT‑PCR Buffer: 12.5 µL

SECTION: Real-time RT-PCR
16.2. 25X RT‑PCR Enzyme Mix: 1 µL

SECTION: Real-time RT-PCR
16.3. CDC-based SARS-CoV-2 N1 primers and probe (FAM dye) are used: 2 µL

SECTION: Real-time RT-PCR
16.4. The VetMAX™ Xeno™ Internal Positive Control (IPC) - LIZ™ Assay (Cy5): 1 µL

SECTION: Real-time RT-PCR
16.5. VetMAX™ Xeno™ Internal Positive Control DNA: 1 µL

SECTION: Real-time RT-PCR
17. Add 20 µLof Master Mix to each well/tube used for RT-PCR 

SECTION: Real-time RT-PCR
16.6. DNase/RNase-free distilled water: 2.5 µL

SECTION: Real-time RT-PCR
18. Add 5 µLRNA templates pooled samples, Negative template control (NTC) such as, DNase/RNase-free distilled Water, and positive control. 

SECTION: Real-time RT-PCR
19. Program real time thermocycler 
SECTION: Real-time RT-PCR
19.1. 48oC for 10 minutes. 
95 oC for 10 minutes.
40 cycles of
95oC for 15 seconds.
60oC for 45 seconds.
SECTION: 8.	Data analysis.
20. Check Ct value of Xeno spiked control for each sample to see if any inhibition occurs