Sep 15, 2025

Public workspaceIsolation and qPCR for S. Typhi detection from River Water Samples

PLOS Neglected Tropical Diseases
DOI
https://dx.doi.org/10.17504/protocols.io.x54v95ym1l3e/v1
  • Nicholas Feasey1,2,3,
  • Nicola Elviss4,
  • Jonathan Rigby1,2,5
  • 1Liverpool School of Tropical Medicine;
  • 2Malawi-Liverpool-Wellcome Trust Programme;
  • 3St. Andrews University;
  • 4UK Health Security Agency;
  • 5Imperial College London
  • ERST MLW LSTM
Jonathan Rigby
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DOI: https://dx.doi.org/10.17504/protocols.io.x54v95ym1l3e/v1
External link: https://doi.org/10.1371/journal.pntd.0012413
Protocol Citation: Nicholas Feasey, Nicola Elviss, Jonathan Rigby 2025. Isolation and qPCR for S. Typhi detection from River Water Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v95ym1l3e/v1
Manuscript citation:
Rigby J, Wilson CN, Zuza A, Diness Y, Mkwanda C, Tonthola K, Kanjerwa O, Salifu C, Pearse O, Msefula C, Perez-Sepulveda BM, Hinton JC, Nair S, Elviss N, Beale MA, Musicha P, Feasey NA (2025) Diversity of Salmonella enterica isolates from urban river and sewage water in Blantyre, Malawi. PLOS Neglected Tropical Diseases 19(9). doi: 10.1371/journal.pntd.0012413
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 29, 2025
Last Modified: September 15, 2025
Protocol Integer ID: 223546
Keywords: salmonella enterica serovar typhi, river water samples this protocol, salmonella, dna extraction, water surveillance, river water sample, validated methodology for water surveillance, enterica serovar typhi, qpcr, time pcr, laboratory processing, moore swab collection, detailed steps for grab sample, molecular confirmation
Funders Acknowledgements:
Bill and Melinda Gates Foundation
Grant ID: INV-008749
Abstract
This protocol outlines a validated methodology for water surveillance of Salmonella enterica serovar Typhi (S. Typhi) through optimised field sampling, selective culture, and confirmatory real-time PCR. It includes detailed steps for grab sample and Moore swab collection, laboratory processing, DNA extraction, and molecular confirmation.
Guidelines
Current guidelines recommended by the Typhoid Environmental Surveillance Consortium can be found at https://www.typhoides.org/ under protocols.
Materials
- Sterile 500 mL or 1 L Nalgene bottles
- Moore swabs (6" x 48", or 15 cm x 120 cm, sterile gauze, folded 8-ply and tied with fishing line)
- Sterile Whirl-Pak bags
- GPS-enabled tablet or field notebook
- Multiprobe water meter (turbidity, pH, temperature, conductivity)
- PPE: gloves, masks, boots
- Cooler box with ice packs (2–8°C transport)
- 70% ethanol for hand and surface sanitation
- Sample ID labels and collection forms (paper/digital)

AB
Distilled Water1,000 mL
Ox Bile (Ref. NCM0240A; Neogen, Manchester, UK)20 g
Dextrose (Ref. NCM0241A; Neogen)5 g
Peptone from gelatin, pancreatic digest (Ref. 70176, Merck, Hertfordshire, UK)10 g
Sodium phosphate dibasic dihydrate (Ref. 71643, Merck)8 g
Potassium dihydrogen phosphate (Ref. NIST200B, Merck)2 g
Table 3: Recipe for Bile- Broth:

AB
Distilled Water1,000 mL
Selenite Broth Base (Ref. CM0395, Oxoid, Basingstoke, UK)38 g
Sodium Biselenite (Ref. LP0121, Oxoid)8 g
Table 4: Recipe for Double Strength Selenite F broth

Troubleshooting
Step 1: Site Selection and Metadata Logging
Use GIS-informed mapping to identify candidate water bodies (streams, rivers, drainage).
Record GPS coordinates and environmental context for each site.
Assess for accessibility and safety; log human or animal activity and environmental conditions.
Complete the site metadata form with:

  • Date/time
  • Weather conditions
  • Recent rainfall
  • Visible contamination
  • Sample type(s) deployed
  • Field team ID
Step 2: Grab Sample Collection
Don appropriate PPE.
Submerge sterile Nalgene bottle ~30 cm below the surface, upstream of your body position.
Collect 1 litre of water.
Seal bottle.
Change gloves and clean surfaces with 80% ethanol.
Affix sample label.
Log sample ID, time, temperature, turbidity, pH, and conductivity.
Step 3: Moore Swab Preparation and Deployment
Prepare swab by folding sterile gauze in a pleated/accordion fashion.
Tie center securely with sterile fishing line (1–2 m retrieval length).
Further instructions can be found at:
Construction of a Moore's Swap - Typhoid ES Consortium
or, Sikorski and Levine, 2020
- See Protocol References at the end of this document
Place swab into mid-stream using line secured to a stable structure (e.g., bridge, tree).
Deploy for 48–72 hours.
Retrieve place directly into sterile Whirl-Pak bag.
Change gloves and clean surfaces with 80% ethanol.
Label bag and log:

  • Deployment duration
  • Retrieval time
  • Any swab loss events

Step 4: Sample Transport and Storage
Place all samples in cooler boxes at 2–8°C.
Transport to lab within 3 hours ideally, 6–8 hours maximum.
If delay to processing is unavoidable, store at 4°C and process within 24 hours.
Record condition on arrival and any temperature deviations.
Step 5: Pre-enrichment – Primary Broth Incubation
For water: filter as much of the sample as possible through 0.45 µm membrane.

- If filters block, change the filter up to 5 membranes
- Stop filtering after 1 hour per sample
- Any water that was not filtered, record volume remaining

Place samples into culture media:

  1. Resuspend filter in 10 mL 2% bile- broth
  2. For Moore swabs: directly submerge whole swab into 250 mL 2% bile- broth.

Incubate at 37 ± 2 °C for 18–24 h.
Step 6: Selective Enrichment – Secondary Broth
Aseptically transfer 5 mL of incubated bile- broth into 5 mL of double strength Selenite F broth.
Incubate at 41 ± 2 °C for 12–18 h (static incubation).
Label all broths with corresponding sample ID and time.
Step 7: Selective Plating on mCASE Agar
Plate the culture media onto modified Chromogenic Agar Salmonella esterase (mCASE) media:

  1. Streak for single colonies
  2. Spread plate at 1:10 dilution
  3. Spread plate at 1:100 dilution

Incubate at 37°C for 18–24 h.
Observe for blue-green colonies (indicative of S. Typhi).
Pick presumptive colonies for molecular confirmation.
Step 8: DNA Extraction
DNA extraction was performed on isolates using the boilate, or thermal lysis, method
Add 500 uL of nuclease free water to a 1.5 mL microcentrifuge tube
Pick 1 to 2 colonies and emulsify into the tube
Heat at 96 °C for 10 minutes
Centrifuge at full speed for 5 seconds to remove condensation
Step 9: Real-Time PCR Confirmation (Triplex Assay)
Prepare reaction mix (25 µL total volume):

ABCDEF
Reagent Working ConcentrationVolume of Working ConcFinal ConcentrationSeqeunce of Primer/ProbeReferences
Takyon Blue 2x Master Mix with ROX 2x12.5 μL1x-
ttr Forward Primer20 μM0.25 μL200 nMCTCACCAGGAGATTACAACATGG(Hopkins et al., 2009)
ttr Reverse Primer20 μM0.25 μL200 nMAGCTCAGACCAAAAGTGACCATC(Hopkins et al., 2009)
ttr Probe (FAM - BHQ1)5 μM0.5 μL100 nMCACCGACGGCGAGACCGACTTT(Hopkins et al., 2009)
tviB Forward Primer20 μM0.5 μL400 nMTGTGGTAAAGGAACTCGGTAAA(Nair et al., 2019)
tviB Reverse Primer20 μM0.5 μL400 nMGACTTCCGATACCGGGATAATG(Nair et al., 2019)
tviB Probe (TET-BHQ1)5 μM1 μL200 nMTGGATGCCGAAGAGGTAAGACGAGA(Nair et al., 2019)
staG Forward Primer20 μM0.5 μL400 nMCGCGAAGTCAGAGTCGACATAG(Nga et al., 2010)
staG Reverse Primer20 μM0.5 μL400 nMAAGACCTCAACGCCGATCAC(Nga et al., 2010)
staG Probe (Cy5 - BHQ2)5 μM1 μL200 nMCATTTGTTCTGGAGCAGGCTGACGG(Nga et al., 2010)
Nuclease Free Water-2.5 μL--
Total Working Volume-20 μL--
DNA Template Volume-5 μL--
Table 1: qPCR Master Mix for S. Typhi detection
Cycling Conditions:

ABCD
StageTemperatureTimeCycles
Hold Stage95 °C5 Minutes1
Denaturation95 °C30 Seconds40
Annealing60 °C30 Seconds
Extension72 °C10 Seconds
Ramp up Speed2.05 °C/s
Reaction Volume25 μL
Table 2: Thermocycling Conditions
Step 10: Data Analysis and Interpretation
Positive sample = amplification of all three targets (ttr, tviB, staG) with Ct < 38.
Include controls:

  1. NTC
  2. qPCR Negative
  3. Positive control

Record Ct values, amplification plots, and annotate inconclusive or single-target results for re-testing.
Archive
All colonies of appropriate morphology and with the ttr gene should be sub-cultured onto fresh mCASE agar
Incubate at 37°C for 18–24 h.
All pure growth should be harvested and placed into Microbank Cryopreservation Beads
  • If culture is not pure, sub-culture until pure
All suspected S. Typhi (positive amplification for ttr, tviB and staG) should be archived separately from the Non-Typhoidal Salmonella isolates
Samples are stored at Ultra-Low Temperature (-80 °C), ensuring minimal freeze-thawing to ensure longevity.
Extraction for Whole Genome Sequencing
All isolates selected for whole genome sequencing will need to be retrieved from Cryobeads
Take one beads from the tube, and place onto a non-selective agar
Make a pool with the bead, and then streak the plates as normal for single colonies
Incubate at 37°C for 18–24 h.
Check for purity by taking one half of a colony for qPCR and subculture the other half (see above sections).
When selected isolates are pure, a 1 μL loop of bacterial growth from nutrient agar and inoculating 1.5 mL of nutrient broth, which was incubated at 37 ± 1°C for 18 to 20 hours
After incubation, samples were heat inactivated at 95 ± 2°C for 10 minutes
700 μL transferred into a deep 96-well plate
centrifuged for 20 minutes at 2,500 x g
The supernatant was discarded and replaced with 220 μL of ATL cell lysis buffer and 20 μL proteinase K
incubated at 60 ± 5 °C for 30 minutes
4 μL of RNase was added and incubated for 15 minutes at 37 ± 1°C
The plate was loaded onto the QiaSymphony
a DSP Virus/Pathogen mini-Kit was used with the default extraction profile on the machine
Yield and purity of each genomic DNA sample after extraction was determined using the Qubit 1x dsDNA Broad Range Assay kit
Protocol references
To develop and optimise methods for the detection and isolation of Salmonella Typhi from the environment.
Rigby, J. (Author). 2022; Student thesis: Doctoral thesis. LSTM Online Archive: https://archive.lstmed.ac.uk/22109/

Rigby, J., et al. (2022). "Optimized methods for detecting Salmonella Typhi in the environment using validated field sampling, culture and confirmatory molecular approaches." J Appl Microbiol 132(2): 1503-1517.

Sikorski, M. J. and M. M. Levine (2020). "Reviving the "Moore Swab": a Classic Environmental Surveillance Tool Involving Filtration of Flowing Surface Water and Sewage Water To Recover Typhoidal Salmonella Bacteria." Applied and environmental microbiology 86(13).

Construction of a Moore Swab: https://www.protocols.io/view/construction-of-a-moore-swab-5jyl85237l2w/v1