Jul 19, 2024

qPCR based multipathogen detection for Salmonella Paratyphi "A" and Vibrio cholerae from wastewater samples

DOI
https://dx.doi.org/10.17504/protocols.io.6qpvr8m8plmk/v1
  • 1Wellcome Trust Research Laboratory, Christian Medical College Vellore, India;
  • 2Department of Community Health and Development, Christian Medical College Vellore, India
Dilip Abraham
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DOI: https://dx.doi.org/10.17504/protocols.io.6qpvr8m8plmk/v1
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 19, 2024
Protocol  Integer ID: 103282
Keywords: qPCR, Environmental surveillance, Wastewater samples, Salmonella Paratyphi A, Vibrio cholerae, multipathogen detection for salmonella paratyphi, different multiplex pcr assay, using multiplex qpcr assay, multiplex qpcr assays from tna, qpcr assay, vibrio cholerae from wastewater sample, vibrio cholerae gene target, salmonella paratyphi, time pcr assay, vibrio cholerae, validation of the primer probe combination, qpcr, primer probe combination, pcr, based multipathogen detection, wastewater sample
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 Targets
CREATED BY
Dilip Abraham


Materials
  1. QuantStudio7 Flex machine
  2. Platinum™ Quantitative PCR SuperMix-UDGThermo FisherCatalog #11730025
  3. qPCR DNA Extraction and Inhibition Control CY5-QXL670EurogentecCatalog #RT-SPCC-Q02
  4. Nuclease-free water AmbionCatalog #AM9932
  5. Primers and Probes (Sigma/IDT)(detailed in protocol)
  6. Positive Controls: gBlocks gene fragments (IDT) (detailed in protocol)
  7. MicroAmp‱ Optical 96-Well Reaction Plate with Barcode Thermofisher Catalog #4306737
  8. MicroAmp‱ Optical Adhesive Film Thermofisher Catalog # 4311971
  9. 1.7 mL MaxyClear Snaplock Microcentrifuge Tube Axygen Catalog #MCT-175-C
  10. Finnpipette F1 100 to 1000 μL Thermo Fisher Catalog #4641100N
  11. Finnpipette F1 20 to 200 μL Thermo Fisher Catalog #4641080N
  12. Finnpipette F1 2 to 20 μL Thermo Fisher Catalog #4641060N
  13. Finnpipette F1 0.2 to 2 μL Thermo Fisher Catalog #4641010N
  14. ART Barrier Specialty Pipette tips 1000 μL Thermo Fisher Catalog #2279-05PK
  15. ART Barrier Specialty Pipette tips 200 μL Thermo Fisher Catalog #2069-05PK
  16. ART Barrier Specialty Pipette tips 20 μL Thermo Fisher Catalog #2149P-05PK
  17. ART Barrier Specialty Pipette tips 10 μL Thermo Fisher Catalog #2139-05PK
  18. 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
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


ABC
TARGETPRIMERS SEQUENCES 5' TO 3'
ctxAForwardTCCGGAGCATAGAGCTTGGA
ReverseTCGATGATCTTGGAGCATTCC
Probe[CY5] - CCGTGGATTCATCATGCACCGC - [BHQ2]
SPA2308ForwardACGATGATGACTGATTTATCGAAC
ReverseTGAAAAGATATCTCTCAGAGCTGG
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


ABC
TARGETPRIMERSSEQUENCES 5' TO 3'
HF183Forward ATCATGAGTTCACA GTCCG
ReverseCTTCCTCTCAGAACCCCTATCC
Probe [FAM] - CTAATGGAACGCATCCC - [BHQ1]
wbfO139Forward AGAAGCCAGTCGCAGTAAAG
ReverseTCGCCATCTTCCAGCATAAA
Probe [TAMRA] - TGGTGGTACAGCTTAGCCGCATTA - [BHQ2]
tcpA classicForward GCGTAATGCAGCAGCTAATAAA
ReverseTATGGGAACATATCACCGACAC
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


ABC
TARGETPRIMERSSEQUENCES 5' TO 3'
OmpWForwardTCAATGATAGCTGGTTCCTCAAC
ReverseCGATGATAAATACCCAAGCATTGA
Probe[JOE]_TGGTATGCCAATATTGAAACAACG_[BHQ1]
wbeO1Forward GTTGAGAAGGGCGGTCTAATAA
ReverseTGTCTGGTACTTGAGTTGGTAAG
Probe[FAM] - TGCCTCAGCAATGGA - [BHQ1]
tcpA EltorForwardATCCTTTCACTGGTACAGCTATG
ReverseGTCAAGCCACCGACTGTAAT
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
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
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
Positive control: gBlocks gene fragments corresponding to each gene target is included in qPCR assay to use as positive control.


ABCD
gBlock geneSequence(5'-3')Accession No:bp size
SPA2308ACGATGATGACTGATTTATCGAACAACGACTCTCCCATACAATTTCATTCTTATTGAGAATGCGCTTATGTAATTTATACCCCAGCTCTGAGAGATATCTTTTCAFM200053.1105
ctxATCCGGAGCATAGAGCTTGGAGGGAAGAGCCGTGGATTCATCATGCACCGCCGGGTTGTGGGAATGCTCCAAGATCATCGAAF463401.180
HF183GGGATCATGAGTTCACATGTCCGCATGATTAAAGGTATTTTCCGGTAGACGAT GGGGATGCGTTCCATTAGATAGTAGGCGGGGTAACGGCCCACCTAGTCAACG ATGGATAGGGGTTCTGAGAGGAAGGTCMT464394.1132
wbfO139AGAAGCCAGTCGCAGTAAAGCACTAGGGCGCATGGTGGTACAGCTTAGCCGCATTATGCGAGATGAGCCGGGTGCGGATTTTATGCTGGAAGATGGCGAAB012956.199
tcpA classicGCGTAATGCAGCAGCTAATAAAGCATTTGCAATTTCAGTGGATGGTCTGACACAGGCTCAATGCAAGACACTTATTACCAGTGTCGGTGATATGTTCCCATAM33514.1102
OmpWTCAATGATAGCTGGTTCCTCAACGCTTCTGTGTGGTATGCCAATATTGAAACAACGGCAACCTACAAAGCAGGTGCAGATGCCAAATCCACGGATGTTGAAATCAATCCTTGGGTATTTATGATCGX51948 modified126
wbeO1GTTGAGAAGGGCGGTCTAATAACACCTAAAGAGTTTGCAGAGAAGCTTGCCTCAGCAATGGATAAGGCTCTTGTACGCTTACCAACTCAAGTACCAGACAKC152957.1100
tcpA El-TorATCCTTTCACTGGTACAGCTATGGGGATTTTCTCATTTCCACGAAACTCTGCAGCGAATAAAGCATTCGCAATTACAGTCGGTGGCTTGACKP187623.191
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
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.

AB
REAGENTVOLUME FOR 1 REACTION (μL)
UDG Mix 12.5
MgCl21
Rox dye0.05
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
NFW5.95
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.


AB
REAGENTVOLUME FOR 1 REACTION (μL)
UDG Mix12.5
MgCl21
ROX dye0.05
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
NFW2.2
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.


AB
REAGENTVOLUME FOR 1 REACTION (μL)
UDG Mix 12.5
MgCl21
ROX dye0.05
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
NFW4.7
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.


ABC
Step ( Hold Stage) Temperature Time
Reverse Transcription50°C2 MIN
PCR initial heat activation 95°C2 MIN
2-step cycling (40 cycles)
Denaturation95°C15 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 fragments
CREATED BY
Jonathan Rigby

Protocol references
1] Kahler AM, Haley BJ, Chen A, Mull BJ, Tarr CL, Turnsek M, Katz LS, Humphrys MS, Derado G, Freeman N, Boncy J, Colwell RR, Huq A, Hill VR. Environmental surveillance for toxigenic Vibrio cholerae in surface waters of Haiti. Am J Trop Med Hyg. 2015 Jan;92(1):118-25. doi: 10.4269/ajtmh.13-0601. Epub 2014 Nov 10. PMID: 25385860; PMCID: PMC4347365. doi: 10.4269/ajtmh.13-0601

2] Alejandro Garrido-Maestu, María-José Chapela, Elvira Peñaranda, Juan M. Vieites, Ana G. Cabado,
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.

3] David R. Greig, Tadgh J. Hickey, Megan D. Boxall, Hafsah Begum, Amy Gentle, Claire Jenkins, Marie A. Chattaway,A real-time multiplex PCR for the identification and typing of Vibrio cholerae,Diagnostic Microbiology and Infectious Disease,Volume 90, Issue 3,2018,Pages 171-176,ISSN 0732-8893, https://doi.org/10.1016/j.diagmicrobio.2017.11.017.

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.

5] https://www.eurogentec.com/assets/b2503b94-10d4-4572-b1fd-95969baeccc3/tds-en-rt-spcc-q-qpcr-dna-extraction-and-inhibition-control-cy5-qxl670.pdf

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