TITLE: qPCR based multipathogen detection for SARS-CoV-2, CrAssphage and Hepatitis E virus from wastewater samples. 
AUTHORS: Dilip Abraham, Blossom Benny, Nirmal Kumar, Karthikeyan Govindan, Venkata Raghava Mohan

[DESCRIPTION]
This protocol outlines the steps for detecting multiple viral gene targets for the pathogens SARS-CoV-2, CrAssphage and HEV using a qPCR assay. Total Nucleic Acid (TNA) extracted from environmental samples (wastewater) is used for testing. 

Key Steps and Considerations:
qPCR Amplification Cycles:
Follow the outlined cycling conditions for optimal amplification of target viral RNA.
 2. Target Detection:
Detect SARS-CoV-2 and HEV using specific primers and probes designed for these targets.


[BEFORE_START]
Fluorescent Dye Selection:
Ensure the chosen fluorescent dyes are compatible with the qPCR machine being used.
If using different dyes than those specified in the protocol, verify their excitation/emission spectra to prevent overlap and avoid crossover between targets.
Calibration of the qPCR machine may be necessary if using alternative fluorescent dyes to ensure accurate detection and measurement.

[GUIDELINES]
This protocol describes qPCR based detection of SARS-CoV-2 and HEV gene targets. Additionally, CrAssphage, an indicator of human fecal contamination, and MS2, serving as an external control, are included in the qPCR assays. Due to the similarity in cycling conditions, these assays can be conducted simultaneously for a sample as two distinct panels.

The positive controls (PCs) used here are linear oligos (gBlocks) that have been used in generating standard curves. Ideally the concentration of PCs used should yield Ct values that fall in the linear phase of the amplification curve.


[STEPS]
SECTION: Primer-Probe Panel
1. The following primers and probes are employed for the detection of SARS-CoV-2, CrAssphage and HEV gene targets.
SECTION: Primer-Probe Reconstitution
2. To reconstitute the lyophilized primers/probes use the nmole information on the specification sheet received with the primers and probes.
SECTION: Primer-Probe Reconstitution
2.1. Multiply nmole value by 10 to get the required volume of Nuclease Free Water (NFW) needed to reconstitute the lyophilized primer/probes.
e.g. For a  primer with 30 nmoles, to make 100 micromolar (µM) stock solution:
30nmol x 10 = 300 µL   of NFW (Nuclease free water) to make 100 micromolar (µM)   stock solution.
SECTION: Primer-Probe Reconstitution
2.2. Add the required volume of NFW,  pulse vortex and spin down. This is the primer / probe stock with 
100 micromolar (µM)    concentration.
SECTION: Primer-Probe Reconstitution
2.3. Store at -20 °C for long term storage.

SECTION: Primer-Probe Dilution
3. Using the  100 micromolar (µM)  stock, prepare a 20 micromolar (µM)  working stock for each primer/probe.
SECTION: Primer-Probe Dilution
3.1. In a fresh tube add 20 µL of 100 micromolar (µM) primer stock  and 80 µL of NFW to make 100 µL of 20 micromolar (µM)  working primer/probe.
SECTION: Primer-Probe Dilution
3.2. Store at 4 °C for frequent usage or -20 °C for long term storage.

SECTION: Preparation of PCR reaction mix
5. Thaw qPCR reagents and samples on ice and briefly spin it down. Do not vortex the AgPath master mix, mix by flicking or pipetting. 
SECTION: Preparation of PCR reaction mix
5.1. SARS-CoV-2/CrAssphage panel 

Prepare the master mix as follows for the number of samples, positive and negative controls, NTC and one extra reaction to account for any pipetting error.

 

SECTION: Primer-Probe Panel
1.2.     The following set of primer-probes are used for the detection of HEV and MS2 targets.


 


SECTION: Primer-Probe Panel
1.1. The following set of primer-probes are used for the detection of SARS-CoV2 and CrAssphage gene targets.

 


SECTION: Preparation of PCR reaction mix
5.2. Hepatitis-E Virus (HEV) and MS2 bacteriophage panel

Prepare the master mix as follows for the number of samples, positive and negative controls, NTC and one extra reaction to account for any pipetting error.

 
HEV and MS2 PCR-  Total volume of master mix to add will be 17 µL 
SECTION: qPCR Controls
4. Controls to be used when performing qPCR assay.
SECTION: Preparation of PCR reaction mix
5.3. Dispense 17 µL of master mix per reaction into the wells of a standard 96-well PCR plate on ice.
Add 3 µL of sample TNA (Total Nucleic Acid). Mix well my pipetting. 
Seal the plate with a roller sealer and then centrifuge the plate for 1 min at 2000g.
SECTION: qPCR Controls
4.1. Positive control: gBlocks gene fragments corresponding to each gene target is included in
PCR panels to use as positive control in pre-defined concentrations.


 

SECTION: qPCR Controls
4.2. Negative control: 3 µL  of extraction blank of each batch of extraction.
NTC: Master mix alone used for no template control.

SECTION: Thermocycler conditions/ program
8. Set up the real time PCR conditions as follows:

 

6. Load the plate into the Quantstudio7 flex instrument after properly starting it up. Open QS7 software, then select - "New experiment set up".
SECTION: Thermocycler conditions/ program
8.1. Click - "Run" to start the qPCR.
SECTION: Thermocycler conditions/ program
8.2. Once the run is complete, adjust the thresholds and baseline if any abnormal baseline at the start or at the end is observed, which may lead to a false-positive curve. Verify if the PC is within the range using defined Ct values chosen from running the standards.
SECTION: Thermocycler conditions/ program
8.3. Export the result to excel/csv file. Upload both run and csv files to Dropbox/OneDrive for backup.
7. Set up the experiment properties with 96-well block, TaqMan reagents,0.2ml PCR plate and
standard run. Define sample ID and define the targets as described for respective PCR panels.
Assign targets and sample ID to each well.
SECTION: Thermocycler conditions/ program
9. The threshold for each target can be set such that the PC for that target falls within the pre-defined range obtained with the standard curves. 

The sample is considered positive if the amplification curve is appropriate and the Ct value falls below the defined cut-off thresholds for each target.

A separate protocol, provided in the Typhoid ES workspace, serves as an example and can be followed to generate Ct cut-off values: