Open BioRad CFX Manager Program.

Setup the proper thermocycler conditions:

Set up well plate template:

• Load the appropriate fluorophores into the appropriate wells (FAM is used for this assay).
• Select for designated controls (no template controls, negative, and positive), standards (with concentrations), and unknowns (samples).
• Name wells for samples accordingly.

Ensure that assay mix is prepared in advance for both N1 and BRSV assays (primers and probes mixed at appropriate concentrations and stored for future use at -20°C , see Appendix I for details).

Ensure that standard curve aliquots have been prepared for SARS-CoV-2 (see Appendix II for details).

Spray surface of hood and wipe pipettes with 10% bleach. Let dry, and repeat with 70% ethanol. Wipe off carefully.

Retrieve RNA from wastewater samples and thaw on ice.

Thaw the reagents from Invitrogen™, primers, and probes at Room temperature .

Prepare spreadsheet list of all samples to run to set up PCR template and to establish volumes needed for each reagent.

Place all reagents on ice, including molecular biological grade water aliquot.

Confirm hood light is off to prevent denaturation of probes before starting mixture preparation.

Prepare two 1.7 mL microcentrifuge tubes labeled "N" and "B". Create the master mixes for each assay by mixing components under a clean hood in the order written in the tables below. Utilize the spreadsheet template to calculate the total volume of each component needed for the number of samples to be run.

A	B	C	D
N1	N (μL/well)	Initial Concentration	Final Concentration / Well
Molecular biology grade water	8.5 μL	–	--
4X TaqPath Master Mix	5.0 μL	4X	1X
IDT N1 Primer and Probe Mix	1.5 μL	13.3X	1X
Primers	--	4 μM	300 nM
Probe	--	2 μM	150 nM
Template RNA	5 μL	–	–
Total volume	20.0 μL	–	--

N Master Mix

A	B	C	D
BRSV	B (μL/well)	Initial Concentration	Final Concentration / Well
Molecular biology grade water	8.0 μL	–	--
4X TaqPath Master Mix	5.0 μL	4X	1X
BRSV Primer and Probe Mix	2.0 μL	10X	1X
Primers	--	4 μM	400 nM
Probe	--	2 μM	200 nM
Template RNA	5 μL	–	–
Total volume	20.0 μL	–	--

B Master Mix

Vortex both N and B mixes for 5 seconds, then spin down for 5 seconds.

Aliquot 15µL of the master mix to each designated well on the PCR plate. Change pipette tips between wells and dispose used pipette tips into trash.

Move the PCR plate to the lab’s PCR bench for adding template. Do not add template in the hood.

Spray surface of PCR bench and wipe pipettes with 10% bleach and then repeat with 70% ethanol. Wipe off carefully.

Retrieve thawed RNA from wastewater samples. Vortex briefly and spin down.

Add 5µL of RNA template into the appropriate well on the PCR template, changing pipette tips in between each well.

For the PCR no template control (NTC), add 5µL of molecular grade water.

For the extraction negative control, add 5µL of control produced each time when extracting the RNA.

For positive control for Moore swab samples, add 5µL of diluted inactivated SARS-CoV-2 sample stored in -80°C freezer.

For the standard curve for grab or composite samples, add 5µL of standards to designated wells in order, starting from the number of lowest to highest genomic copies (gc).

A	B	C	D	E
Std G	Std F	Std E	Std D	Std C
10 gc	100 gc	1,000 gc	10,000 gc	100,000 gc

gc = genomic copies

Once plate is completed, seal the wells with adhesive sealing film and spin down for 0h 5m 0s on a balanced 96-well plate centrifuge.

Place samples in Bio-Rad Detection System and start PCR run with the appropriate thermocycling conditions as listed above.

Wipe down the pipette and surface with 10% bleach and then 70% ethanol.

Store leftover RNA samples in the freezer at -20°C .

Export data as an Excel file or CSV file for viewing and analysis.

After viewing results, utilize the following decision tree to determine if results pass QA/QC.

• If both Ct values are present and the Ct difference is less than 2 --> Positive

1. If the lower Ct value is less than 36 --> Quantifiable positive
2. If the lower Ct value is greater than 36 --> Non-quantifiable positive

• If one Ct value is greater than 36 while the other is negative --> Negative
• If one Ct value is less than 36 while the other is negative --> Rerun sample
• If both Ct values are less than 36 and the Ct difference is greater than 2 --> Rerun sample
• If any Ct value of BRSV is negative --> Rerun sample
• For re-rerun results, if both Ct values are present and the difference is greater than 2 --> Positive, average the Ct values. Otherwise, follow the same aforementioned guideline

For each assay, combine primers and probes to create a stock mix with a concentration of 4 μM for each primer and 2μM for the probe.

Example to create 250 μL primer probe mix with 100 μM starting solutions:

• 10 μL Forward Primer (100 μM stock)
• 10 μL Reverse Primer (100 μM stock)
• 5 μL Probe (100 μM stock)
• 225 μL of microbiological grade H₂O

Vortex the mixture on a benchtop vortex and place into a -20°C freezer until needed for PCR.

The standard curve should be composed of the following and generated as a dilution series:

A	B	C	D	E	F	G
Dilution #	Name	RNA (μL)	Molecular Water (μL)	Final Volume (μL)	gc/μL	gc/5μL
1	B	27	73	100	200000	1000000
2	C	30	270	300	20000	100000
3	D	30	270	300	2000	10000
4	E	30	270	300	200	1000
5	F	30	270	300	20	100
6	G	30	270	300	2	10

gc = genomic copies

Thaw Quantitative Synthetic SARS-CoV-2 RNA on ice. Add 27µL of RNA into 73µL of molecular water to create 100 μL dilution B.

Add 30µL of dilution B into 270µL of molecular water to create 300 μL dilution C. Vortex the mix.

Add 30µL of dilution C into 270µL of molecular water to create 300 μL dilution D. Vortex the mix.

Add 30µL of dilution D into 270µL of molecular water to create 300 μL dilution E. Vortex the mix.

Add 30µL of dilution E into 270µL of molecular water to create 300 μL dilution F. Vortex the mix.

Add 30µL of dilution F into 270µL of molecular water to create 300 μL dilution G. Vortex the mix.

Make 14 μL aliquots for each dilution and save at -20°C freezer. Keep the remaining dilution B in the -70°C freezer for future use.