qPCR based multipathogen detection for SARS-CoV-2, CrAssphage and Hepatitis E virus from wastewater samples.
Dilip Abraham, Venkata Raghava Mohan, Nirmal Kumar, Blossom Benny, Karthikeyan Govindan
Abstract
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.
- 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.
Steps
Primer-Probe Panel
The following primers and probes are employed for the detection of SARS-CoV-2, CrAssphage and HEV gene targets.
The following set of primer-probes are used for the detection of SARS-CoV2 and CrAssphage gene targets.
| A | B | C |
|---|---|---|
| TARGET | PRIMERS | SEQUENCES 5' TO 3' |
| SARS-CoV-2 N1 | Forward | GACCCCAAAATCAGCGAAAT |
| Reverse | TCTGGTTACTGCCAGTTGAATCTG | |
| Probe | [JOE]-ACCCCGCATTACGTTTGGTGGACC-[BHQ1] | |
| SARS-CoV-2 -N2 | Forward | TTACAAACATTGGCCGCAAA |
| Reverse | GCGCGACATTCCGAAGAA | |
| Probe | [TAMRA]-ACAATTTGCCCCCAGCGCTTCAG-[BHQ2] | |
| CrAssphage | Forward | CAGAAGTACAAACTCCTAAAAAACGTAGAG |
| Reverse | GATGACCAATAAACAAGCCATTAGC | |
| Probe | [FAM]-AATAACGATTTACGTGATGTAAC-[MGB] |
Table 1: Primers and Probes for SARS-CoV-2 and CrAssphage. Fluorescent dyes and quenchers are shown in square brackets.
The following set of primer-probes are used for the detection of HEV and MS2 targets.
| A | B | C |
|---|---|---|
| TARGET | PRIMERS | SEQUENCES 5' TO 3' |
| HEV | Forward | GGTGGTTTCTGGGGTGAC |
| Reverse | AGGGGTTGGTTGGATGAA | |
| Probe | [FAM]-TGATTCTCAGCCCTTCGC-[MGB] | |
| MS2 | Forward | TGGCACTACCCCTCTCCGTATTCAC |
| Reverse | GTACGGGCGACCCCACGATGAC | |
| Probe | [TAMRA]-CACATCGATAGATCAAGGTGCC-[BHQ2] |
Table 2: Primers and Probes for HEV and MS2. Fluorescent dyes and quenchers are shown in square brackets.
Primer-Probe Reconstitution
To reconstitute the lyophilized primers/probes use the nmole information on the specification sheet received with the primers and probes.
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 100micromolar (µM) stock solution:
30nmol x 10 =300µL of NFW (Nuclease free water) to make100micromolar (µM) stock solution.
Add the required volume of NFW, pulse vortex and spin down. This is the primer / probe stock with
100micromolar (µM) concentration.
Store at -20°C for long term storage.
Primer-Probe Dilution
Using the 100micromolar (µM) stock, prepare a 20micromolar (µM) working stock for each primer/probe.
In a fresh tube add 20µL of 100micromolar (µM) primer stock and 80µL of NFW to make 100µL of 20micromolar (µM) working primer/probe.
Store at 4°C for frequent usage or -20°C for long term storage.
qPCR Controls
Controls to be used when performing qPCR assay.
Positive control: gBlocks gene fragments corresponding to each gene target is included in
PCR panels to use as positive control in pre-defined concentrations.
| A | B | C |
|---|---|---|
| SARS-CoV-2 N1 | TTCATCTAAACGAACAAACTAAAATGTCTGATAATGGACCCCAAAATCAGCGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCCAAGGTTTACCCAATAATACT | 171 |
| SARS-CoV-2 N2 | TGGGGACCAGGAACTAATCAGACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCCAGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACCTTCGGGAACGTGGTTGACCT ACACAGGTGCCATCAAATTG | 161 |
| HEV | GGTGGTTTCTGGGGTGACCGGGTTGATTCTCAGCCCTTCGCAATCCCCTATATTCATCCAACCAACCCCT | 70 |
| CrAssphage | CAGAAGTACAAACTCCTAAAAAACGTAGAGGTAGAGGTATTAATAACGATTTACGTGATGTAACTCGTAAAAAGTTTGATGAACGTACTGATTGTAATAAAGCTAATGGCTTGTTTATTGGTCATC | 126 |
Table 3: Sequences used for gBlocks gene fragments. Sequences for the targets SARS-CoV-2 N1 and N2 is taken from SARS-CoV-2 reference genome NC_045512.2) and the HEV sequence is taken from the HEV virus complete genome sequence(MN401238.1). CrAssphage reference genome (MK415410.1).
Negative control: 3µL of extraction blank of each batch of extraction.
NTC : Master mix alone used for no template control.
Preparation of PCR reaction mix
Thaw qPCR reagents and samples on ice and briefly spin it down. Do not vortex the AgPath master mix, mix by flicking or pipetting.
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.
| A | B |
|---|---|
| Ag Path master mix | 10 |
| 25x Enzyme mix | 0.8 |
| N1 F primer | 0.25 |
| N2 R primer | 0.25 |
| N1 probe | 0.125 |
| N2 F primer | 0.25 |
| N2 R primer | 0.25 |
| N2 probe | 0.125 |
| CPQ F primer | 0.25 |
| CPQ R primer | 0.25 |
| CPQ probe | 0.125 |
| NFW | 4.325 |
Table 4: SARS-CoV-2/CrAssphage - PCR reaction mix
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.
| A | B |
|---|---|
| Ag Path master mix | 10 |
| 25x Enzyme mix | 0.8 |
| HEV F primer | 0.25 |
| HEV R primer | 0.25 |
| HEV probe | 0.125 |
| MS2 F primer | 0.25 |
| MS2 R primer | 0.25 |
| MS2 probe | 0.125 |
| NFW | 4.95 |
Table 5: HEV/MS2 PCR reaction mix
HEV and MS2 PCR- Total volume of master mix to add will be 17µL
Dispense 17µL of master mix per reaction into the wells of a standard 96-well PCR plate on ice.
Add 3µL 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.
Load the plate into the Quantstudio7 flex instrument after properly starting it up. Open QS7 software, then select - "New experiment set up".
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.
Thermocycler conditions/ program
Set up the real time PCR conditions as follows:
| A | B | C |
|---|---|---|
| Reverse Transcription | 45° C | 20 min |
| PCR initial heat activation | 95° C | 10 min |
| 2-step cycling (40 cycles) | ||
| Denaturation | 95° C | 15 sec |
| Combined annealing/extension | 55° C (data collection step) | 1 min |
Click - "Run" to start the qPCR.
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.
Export the result to excel/csv file. Upload both run and csv files to Dropbox/OneDrive for backup.
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:
