Fecal sample pooling for SARS-CoV-2 real-time RT-PCR screening

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

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.

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.

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

48^oC for 10 minutes.

95^oC for 10 minutes.

40 cycles of

95^oC for 15 seconds.

60^oC for 45 seconds.

Check Ct value of Xeno spiked control for each sample to see if any inhibition occurs