TITLE: Singleplex qPCR for SARS-CoV-2 N1 and BRSV
AUTHORS: Chloe   Svezia, Anh   Nguyen, Jamie   VanTassell, Julia  Raymond, Marlene K Wolfe, Pengbo   Liu, Christine  Moe

[DESCRIPTION]
This protocol describes the procedure to perform RT-qPCR for the detection of the SARS-CoV-2 N gene and a processing control (BRSV) in RNA extracted from wastewater samples. For samples obtained with a Moore swab, results will indicate the presence or absence of each target. For grab or composite wastewater samples, results may be used to estimate the concentration of the target in the original sample.

[STEPS]
SECTION: Preparation
4. 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).

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

SECTION: Preparation
6. Spray surface of hood and wipe pipettes with 10% bleach. Let dry, and repeat with 70% ethanol. Wipe off carefully.
SECTION: Preparation
7. Retrieve RNA from wastewater samples and thaw on ice.
SECTION: Preparation
8. Thaw the reagents from Invitrogen™, primers, and probes at Room temperature .

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

SECTION: Procedure
10. Place all reagents on ice, including molecular biological grade water aliquot.
SECTION: Procedure
11. Confirm hood light is off to prevent denaturation of probes before starting mixture preparation.
SECTION: Procedure
12. 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.
 
        N         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         –         --      
  
 
        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            [1] [2] [3] [4] [5] BRSV Primer and Probe Mix         2.0 μL         10X         1X            [6] [7] [8] [9] [10] Primers         --         4 μM         400 nM            [11] [12] [13] [14] [15] Probe         --         2 μM         200 nM            [16] [17] [18] [19] [20] Template RNA         5 μL         –         –            Total   volume         20.0 μL         –         --      
 
SECTION: Procedure
13. Vortex both N and B mixes for 5 seconds, then spin down for 5 seconds.
SECTION: Procedure
14. Aliquot 15 µL of the master mix to each designated well on the PCR plate. Change pipette tips between wells and dispose used pipette tips into trash.

SECTION: Procedure
15. Move the PCR plate to the lab’s PCR bench for adding template. Do not add template in the hood.
SECTION: Procedure
16. Spray surface of PCR bench and wipe pipettes with 10% bleach and then repeat with 70% ethanol. Wipe off carefully.
SECTION: Procedure
17. Retrieve thawed RNA from wastewater samples. Vortex briefly and spin down.
SECTION: Procedure
18. Add 5 µL of RNA template into the appropriate well on the PCR template, changing pipette tips in between each well.

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

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

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

SECTION: Procedure
22. For the standard curve for grab or composite samples, add 5 µL of standards to designated wells in order, starting from the number of lowest to highest genomic copies (gc).
       Std G         Std F         Std E         Std D         Std C             10 gc         100 gc         1,000 gc         10,000 gc         100,000 gc      
 

SECTION: Procedure
23. Once plate is completed, seal the wells with adhesive sealing film and spin down for 5 min on a balanced 96-well plate centrifuge.

SECTION: Procedure
24. Place samples in Bio-Rad Detection System and start PCR run with the appropriate thermocycling conditions as listed above.
SECTION: Procedure
25. Wipe down the pipette and surface with 10% bleach and then 70% ethanol.
SECTION: Procedure
26. Store leftover RNA samples in the freezer at -20 °C .

SECTION: Appendix I - Primer/Probe Mix
29. 
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. 


SECTION: Software Preparation
1. Open BioRad CFX Manager Program.
SECTION: Software Preparation
2. Setup the proper thermocycler conditions:

   

SECTION: Software Preparation
3. 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.
SECTION: Appendix I - Primer/Probe Mix
30. Vortex the mixture on a benchtop vortex and place into a -20 °C freezer until needed for PCR.

SECTION: Appendix I - Primer/Probe Mix
29.1. 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
SECTION: Appendix II - Standard Curve Preparation
31.  The standard curve should be composed of the following and generated as a dilution series:
       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      
 
SECTION: Appendix II - Standard Curve Preparation
32. Thaw Quantitative Synthetic SARS-CoV-2 RNA on ice. Add 27 µL of RNA into 73 µL of molecular water to create 100 μL dilution B.
 



SECTION: Appendix II - Standard Curve Preparation
33. Add 30 µL of dilution B into 270 µL of molecular water to create 300 μL dilution C. Vortex the mix.

SECTION: Appendix II - Standard Curve Preparation
34. Add 30 µL of dilution C into 270 µL of molecular water to create 300 μL dilution D. Vortex the mix.

SECTION: Appendix II - Standard Curve Preparation
35. Add 30 µL of dilution D into 270 µL of molecular water to create 300 μL dilution E. Vortex the mix.
SECTION: Appendix II - Standard Curve Preparation
36. Add 30 µL of dilution E into 270 µL of molecular water to create 300 μL dilution F. Vortex the mix.
SECTION: Appendix II - Standard Curve Preparation
37. Add 30 µL of dilution F into 270 µL of molecular water to create 300 μL dilution G. Vortex the mix.
SECTION: Appendix II - Standard Curve Preparation
38. 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.

SECTION: Viewing and Saving Data
27. Export data as an Excel file or CSV file for viewing and analysis.
SECTION: Interpreting Results
28. 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
If the lower Ct value is less than 36 --> Quantifiable positive
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