Jul 20, 2024
qPCR based multipathogen detection for Salmonella Paratyphi "A" and Vibrio cholerae from wastewater samples
- Dilip Abraham1,
- Blossom Benny Sam1,
- Nirmal Kumar1,
- Raju Ravi1,
- Venkata Raghava Mohan2
- 1Wellcome Trust Research Laboratory, Christian Medical College Vellore, India;
- 2Department of Community Health and Development, Christian Medical College Vellore, India
Protocol Citation: Dilip Abraham, Blossom Benny Sam, Nirmal Kumar, Raju Ravi, Venkata Raghava Mohan 2024. qPCR based multipathogen detection for Salmonella Paratyphi "A" and Vibrio cholerae from wastewater samples. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8m8plmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 12, 2024
Last Modified: July 20, 2024
Protocol Integer ID: 103282
Keywords: qPCR, Environmental surveillance, Wastewater samples, Salmonella Paratyphi A, Vibrio cholerae
Funders Acknowledgements:
Bill & Melinda Gates Foundation
Grant ID: INV-049093
Bill & Melinda Gates Foundation
Grant ID: INV-048134
Abstract
The following protocol is optimized for the detection of Salmonella Paratyphi A and Vibrio cholerae using multiplex qPCR assays from TNA extracted from environmental samples (wastewater samples). Total Nucleic Acid (TNA) extracted from the pellet particles is used as template in a real-time PCR assay which uses primer-probes specific for Salmonella Paratyphi A or Vibrio cholerae gene targets. Three different multiplex PCR assays were designed following standardization and validation of the primer probe combinations.
Guidelines
This protocol describes qPCR based detection of Salmonella Paratyphi "A" and Vibrio cholerae gene targets in three PCR panels from TNA extraction of processed environmental samples that were spiked with internal controls (SPC EGT control DNA - referenced below) . Additionally, HF183 which serves as a marker of fecal contamination in environmental samples, is included in the qPCR assays. Due to the similarity in cycling conditions, these assays can be conducted simultaneously for a sample as three 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.
Protocol
NAME
Extraction of Total Nucleic Acid from Environmental Samples for the Detection of Bacterial and Viral TargetsCREATED BY
Dilip Abraham
Materials
- QuantStudio7 Flex machine
- Platinum™ Quantitative PCR SuperMix-UDGThermo FisherCatalog #11730025
- qPCR DNA Extraction and Inhibition Control CY5-QXL670EurogentecCatalog #RT-SPCC-Q02
- Nuclease-free water AmbionCatalog #AM9932
- Primers and Probes (Sigma/IDT)(detailed in protocol)
- Positive Controls: gBlocks gene fragments (IDT) (detailed in protocol)
- MicroAmp‱ Optical 96-Well Reaction Plate with Barcode Thermofisher Catalog #4306737
- MicroAmp‱ Optical Adhesive Film Thermofisher Catalog # 4311971
- 1.7 mL MaxyClear Snaplock Microcentrifuge Tube Axygen Catalog #MCT-175-C
- Finnpipette F1 100 to 1000 μL Thermo Fisher Catalog #4641100N
- Finnpipette F1 20 to 200 μL Thermo Fisher Catalog #4641080N
- Finnpipette F1 2 to 20 μL Thermo Fisher Catalog #4641060N
- Finnpipette F1 0.2 to 2 μL Thermo Fisher Catalog #4641010N
- ART Barrier Specialty Pipette tips 1000 μL Thermo Fisher Catalog #2279-05PK
- ART Barrier Specialty Pipette tips 200 μL Thermo Fisher Catalog #2069-05PK
- ART Barrier Specialty Pipette tips 20 μL Thermo Fisher Catalog #2149P-05PK
- ART Barrier Specialty Pipette tips 10 μL Thermo Fisher Catalog #2139-05PK
- Microplate Centrifuge, PCR Plate Spinner VWR‱ Catalog #VWRU89184-610
Before start
Ensure that the fluorescent dye used for TaqMan probes is compatible with the machine being used and properly calibrated for it.
Primer-Probe panel
Primer-Probe panel
The following primers and probes are employed for the detect Salmonella Paratyphi A and Vibrio cholerae specific gene targets.
PCR 1: Salmonella Paratyphi A (SPA2308) and V. cholerae ctxA PCR panel
| A | B | C | |
| TARGET | PRIMERS | SEQUENCES 5' TO 3' | |
| ctxA | Forward | TCCGGAGCATAGAGCTTGGA | |
| Reverse | TCGATGATCTTGGAGCATTCC | ||
| Probe | [CY5] - CCGTGGATTCATCATGCACCGC - [BHQ2] | ||
| SPA2308 | Forward | ACGATGATGACTGATTTATCGAAC | |
| Reverse | TGAAAAGATATCTCTCAGAGCTGG | ||
| Probe | [FAM] - CCCATACAATTTCATTCTTATTGAGAATGCGC - [BHQ1] |
Table1: SPA and ctxA Primer and probe sequences. Fluorescent dyes and quenchers are shown in square brackets.
PCR 2: Vibrio cholerae_1 PCR panel
| A | B | C | |
| TARGET | PRIMERS | SEQUENCES 5' TO 3' | |
| HF183 | Forward | ATCATGAGTTCACA GTCCG | |
| Reverse | CTTCCTCTCAGAACCCCTATCC | ||
| Probe | [FAM] - CTAATGGAACGCATCCC - [BHQ1] | ||
| wbfO139 | Forward | AGAAGCCAGTCGCAGTAAAG | |
| Reverse | TCGCCATCTTCCAGCATAAA | ||
| Probe | [TAMRA] - TGGTGGTACAGCTTAGCCGCATTA - [BHQ2] | ||
| tcpA classic | Forward | GCGTAATGCAGCAGCTAATAAA | |
| Reverse | TATGGGAACATATCACCGACAC | ||
| Probe | [JOE] - ATGGTCTGACACAGGCTCAATGCA - [BHQ1] |
Table2: Vibrio cholerae_1 PCR Primer and probe sequences. Fluorescent dyes and quenchers are shown in square brackets.
PCR 3: Vibrio cholerae_2 PCR panel
| A | B | C | |
| TARGET | PRIMERS | SEQUENCES 5' TO 3' | |
| OmpW | Forward | TCAATGATAGCTGGTTCCTCAAC | |
| Reverse | CGATGATAAATACCCAAGCATTGA | ||
| Probe | [JOE]_TGGTATGCCAATATTGAAACAACG_[BHQ1] | ||
| wbeO1 | Forward | GTTGAGAAGGGCGGTCTAATAA | |
| Reverse | TGTCTGGTACTTGAGTTGGTAAG | ||
| Probe | [FAM] - TGCCTCAGCAATGGA - [BHQ1] | ||
| tcpA Eltor | Forward | ATCCTTTCACTGGTACAGCTATG | |
| Reverse | GTCAAGCCACCGACTGTAAT | ||
| Probe | [TAMRA]- ACGAAACTCTGCAGCGAATAAAGC-[BHQ2] |
Table3: Vibrio cholerae_2 PCR Primer and probe sequences. Fluorescent dyes and quenchers are shown in square brackets.
Primer-probe reconstitution
Primer-probe reconstitution
To reconstitute the lyophilized primers/probes, use the nmole information on the specification sheet received with primers/probes.
Multiply nmol value by 10 to get the required volume of Nuclease Free Water (NFW) needed to reconstitute the lyophilized primer/probes.
Ex: For a primer with 30 nmoles, to make 100 micromolar (µM) stock solution:
30 nmol x 10 = 300 µL of NFW to make 100 micromolar (µM) stock solution.
Add the required volume of nuclease free water, pulse vortex and spin down. This is the primer/probe stock with 100 micromolar (µM) concentration.
Store at -20 °C for long term storage.
Primer-probe dilution
Primer-probe dilution
Prepare a working stock from 100 micromolar (µM) stock solution.
In a fresh tube add 10 µL of 100 micromolar (µM) primer/probe stock and 90 µL of nuclease free water to give 100 µL of 10 micromolar (µM) working primer/probe.
Store at 4 °C for frequent usage or -20 °C for long-term storage.
qPCR controls
qPCR controls
Positive control: gBlocks gene fragments corresponding to each gene target is included in qPCR assay to use as positive control.
| A | B | C | D | |
| gBlock gene | Sequence(5'-3') | Accession No: | bp size | |
| SPA2308 | ACGATGATGACTGATTTATCGAACAACGACTCTCCCATACAATTTCATTCTTATTGAGAATGCGCTTATGTAATTTATACCCCAGCTCTGAGAGATATCTTTTCA | FM200053.1 | 105 | |
| ctxA | TCCGGAGCATAGAGCTTGGAGGGAAGAGCCGTGGATTCATCATGCACCGCCGGGTTGTGGGAATGCTCCAAGATCATCGA | AF463401.1 | 80 | |
| HF183 | GGGATCATGAGTTCACATGTCCGCATGATTAAAGGTATTTTCCGGTAGACGAT GGGGATGCGTTCCATTAGATAGTAGGCGGGGTAACGGCCCACCTAGTCAACG ATGGATAGGGGTTCTGAGAGGAAGGTC | MT464394.1 | 132 | |
| wbfO139 | AGAAGCCAGTCGCAGTAAAGCACTAGGGCGCATGGTGGTACAGCTTAGCCGCATTATGCGAGATGAGCCGGGTGCGGATTTTATGCTGGAAGATGGCGA | AB012956.1 | 99 | |
| tcpA classic | GCGTAATGCAGCAGCTAATAAAGCATTTGCAATTTCAGTGGATGGTCTGACACAGGCTCAATGCAAGACACTTATTACCAGTGTCGGTGATATGTTCCCATA | M33514.1 | 102 | |
| OmpW | TCAATGATAGCTGGTTCCTCAACGCTTCTGTGTGGTATGCCAATATTGAAACAACGGCAACCTACAAAGCAGGTGCAGATGCCAAATCCACGGATGTTGAAATCAATCCTTGGGTATTTATGATCG | X51948 modified | 126 | |
| wbeO1 | GTTGAGAAGGGCGGTCTAATAACACCTAAAGAGTTTGCAGAGAAGCTTGCCTCAGCAATGGATAAGGCTCTTGTACGCTTACCAACTCAAGTACCAGACA | KC152957.1 | 100 | |
| tcpA El-Tor | ATCCTTTCACTGGTACAGCTATGGGGATTTTCTCATTTCCACGAAACTCTGCAGCGAATAAAGCATTCGCAATTACAGTCGGTGGCTTGAC | KP187623.1 | 91 |
Table4: Sequences used for gBlocks gene fragments
Negative control: 3 µL of extraction blank of each batch of extraction.
NTC: Master mix alone used for no template control.
Quantitative PCR
Quantitative PCR
Thaw qPCR reagents and samples on ice and briefly spin it down.
Prepare master mix for each of the 3 primer-probe panels.
[PCR 1] Salmonella Paratyphi A (SPA2308) and ctxA
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 | |
| REAGENT | VOLUME FOR 1 REACTION (μL) | |
| UDG Mix | 12.5 | |
| MgCl2 | 1 | |
| Rox dye | 0.05 | |
| ctxA F Primer (10 µM) | 0.5 | |
| ctxA R Primer (10 µM) | 0.5 | |
| ctxA Probe (10 µM) | 0.25 | |
| SPA2308 F (10 µM) | 0.5 | |
| SPA2308 R (10 µM) | 0.5 | |
| SPA2308 Probe (10 µM) | 0.25 | |
| NFW | 5.95 |
Table5: Mastermix composition for SPA2308 and ctxA gene targets
[PCR 2] Vibrio cholerae_1 PCR
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 | |
| REAGENT | VOLUME FOR 1 REACTION (μL) | |
| UDG Mix | 12.5 | |
| MgCl2 | 1 | |
| ROX dye | 0.05 | |
| HF183 F Primer (10 µM) | 0.5 | |
| HF183 R Primer (10 µM) | 0.5 | |
| HF183 Probe (10 µM) | 0.25 | |
| wbfO139 F Primer (10 µM) | 0.5 | |
| wbfO139 R Primer (10 µM) | 0.5 | |
| wbfO139 Probe (10 µM) | 0.25 | |
| tcpA classic F Primer (10 µM) | 0.5 | |
| tcpA classic R Primer (10 µM) | 0.5 | |
| tcpA classic Probe (10 µM) | 0.25 | |
| SPC (10 X EGT Control Mix) | 2.5 | |
| NFW | 2.2 |
Table6: Mastermix composition for Vibrio cholerae_1 PCR gene targets
Note
SPC is Sample Processing Control, an optimized TaqMan control designed to be used as qPCR DNA extraction and inhibition control. It is detected by Cy5-labelled probe (Cy5-QXL670 Probe) which comes as ready to use primer probe mix.
[PCR 3] Vibrio cholerae_2 PCR
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 | |
| REAGENT | VOLUME FOR 1 REACTION (μL) | |
| UDG Mix | 12.5 | |
| MgCl2 | 1 | |
| ROX dye | 0.05 | |
| OmpW F Primer (10 µM) | 0.5 | |
| OmpW R Primer (10 µM) | 0.5 | |
| OmpW Probe (10 µM) | 0.25 | |
| wbeO1 F Primer (10 µM) | 0.5 | |
| wbeO1 R Primer (10 µM) | 0.5 | |
| wbeO1 Probe (10 µM) | 0.25 | |
| tcpA El-Tor F Primer (10 µM) | 0.5 | |
| tcpA El-Tor R Primer(10µM) | 0.5 | |
| tcpA El-Tor Probe (10 µM) | 0.25 | |
| NFW | 4.7 |
Table7: Mastermix composition for Vibrio cholerae_2 PCR gene targets
For each PCR, aliquot 22 μl of master mix to each required well in a 96-well plate.
Add 3μl of sample, or 3 μl of nuclease free water for negative controls.
Seal the plate with a roller sealer and then centrifuge the plate for 1 min at 2,000g.
Load the plate into Quantstudio7 flex instrument after proper initiation of the instrument. Open QS7 software, then select "New Experiment set up".
Set up the experiment properties with 96-well block, TaqMan reagents, 0.2 ml 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.
Set up the PCR cycling method as described below.
Cycling conditions remain the same for all 3 qPCRs.
| A | B | C | |
| Step ( Hold Stage) | Temperature | Time | |
| Reverse Transcription | 50°C | 2 MIN | |
| PCR initial heat activation | 95°C | 2 MIN | |
| 2-step cycling (40 cycles) | |||
| Denaturation | 95°C | 15 SEC | |
| Combined annealing/extension | 60°C (data collection step) | 30 SEC | |
Start the qPCR by clicking “Run.”
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 the cut-off Ct values chosen from running the standards.
Export the result to excel/csv file and upload both run and csv files.
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:
Protocol
NAME
Generating Ct cut-off values using gBlocks gene fragmentsCREATED BY
Catherine Troman
Protocol references
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25385860; PMCID: PMC4347365. doi: 10.4269/ajtmh.13-0601
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In-house validation of novel multiplex real-time PCR gene combination for the simultaneous detection of the main human pathogenic vibrios (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus),Food Control,
Volume 37,2014,Pages 371-379,ISSN 0956-7135,https://doi.org/10.1016/j.foodcont.2013.09.026.
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4] Nga TV, Karkey A, Dongol S, et al. The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens. BMC Infect Dis. 2010;10:125. Published 2010 May 21. doi:10.1186/1471-2334-10-125.
6] Thong KL, Tham KBL, Ngoi ST, et al. Molecular characterization of Vibrio cholerae O1 El Tor strains in Malaysia revealed genetically diverse variant lineages. Transbound Emerg Dis. 2022;69(4):e693-e703. doi:10.1111/tbed.14368