Dec 16, 2025
Version 4
Extraction and qPCR of Environmental Surveillance samples for the detection of Salmonella Typhi V.4
- Jonathan Rigby1,2,3,
- Catherine Troman4,1,
- Dilip Abraham5,
- Jaspreet Mahindroo1,
- Christopher Uzzell1,
- Jacob John5,
- Venkata Raghava Mohan5,
- Rajan Srinivasan5,
- Nicolette Zhou6,
- Satheesh Nair7,
- Alex Shaw1,
- Nicola Elviss7,
- Nicholas Feasey8,
- John Scott Meschke6,
- Gagandeep Kang5,
- Nick Grassly1
- 1Imperial College London;
- 2Malawi Liverpool Wellcome Trust;
- 3Liverpool School of Tropical Medicine;
- 4UiT The Arctic University of Norway;
- 5Christian Medical College, Vellore, India;
- 6University of Washington;
- 7UK Health Security Agency;
- 8Malawi Liverpool Wellcome Trust Clinical Research Program, Blantyre, Malawi
- Typhoid Environmental Surveillance
Protocol Citation: Jonathan Rigby, Catherine Troman, Dilip Abraham, Jaspreet Mahindroo, Christopher Uzzell, Jacob John, Venkata Raghava Mohan, Rajan Srinivasan, Nicolette Zhou, Satheesh Nair, Alex Shaw, Nicola Elviss, Nicholas Feasey, John Scott Meschke, Gagandeep Kang, Nick Grassly 2025. Extraction and qPCR of Environmental Surveillance samples for the detection of Salmonella Typhi. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5zrw4v1b/v4Version created by Jonathan Rigby
Manuscript citation:
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: December 12, 2025
Last Modified: December 16, 2025
Protocol Integer ID: 234882
Keywords: Salmonella, Typhi, Paratyphi, wastewater, environmental surveillance, detection of salmonella typhi, identifying salmonella typhi, qpcr of environmental surveillance sample, salmonella typhi, qpcr analysis of the sample, dna extraction, stage of dna extraction, qpcr analysis, fluorescence of taqman, qpcr, environmental surveillance sample, human faecal indicator, moore swab, use on environmental sample, fluorescence, fluorescent dye, environmental sample, detection, extraction, dna, typhoid es workspace
Funders Acknowledgements:
Bill and Melinda Gates Foundation
Abstract
The following protocol is for use on environmental samples that have been collected by Moore swab or grab sampling methods and processed up to the stage of DNA extraction. The protocol includes DNA extraction and qPCR analysis of the samples to detect three gene targets for identifying Salmonella Typhi, a human faecal indicator (HF183), and a commercial spike-in control.
The qPCR uses fluorescence of Taqman probes for measuring the product. Care must be taken to ensure that the fluorescent dyes chosen for the probes are suitable for the machine being used or can be calibrated for use on the machine. If the dyes selected are different from those described in the protocol, make sure that their excitation/emission spectra do not overlap to avoid crossover between targets.
Generation of standard curves is mentioned in this protocol but is described in more detail in a separate protocol in the Typhoid ES workspace.
Materials
QIAamp PowerFecal Pro DNA KitQiagenCatalog #51804
Water, nuclease free
qPCR DNA Extraction and Inhibition Control CY5-QXL670EurogentecCatalog #RT-SPCC-Q02
Takyon Low ROX Probe 2x MasterMix dTTP blueEurogentecCatalog #UF-LPMT-B0701
Primers and Probes (detailed in protocol)
gBlocks DNA fragments (detailed in protocol)
qPCR machine
96-well plates for qPCR and plate seals
Vortex with tube adapter
96-well plate spinner
Microcentrifuge
1.5ml tubes
Troubleshooting
DNA Extraction
For the pellet from membrane filtration, resuspend the pellet in 800 µL CD1 from the first step of the DNA extraction protocol, then transfer to the PowerBead tube for homogenisation.
For extraction from Moore swabs, add 800 µL of CD1 to the membrane strips already stored in PowerBead tubes.
If stored in another microcentrifuge tube, pour the zirconia beads from a PowerBead tube included in the Qiagen PowerFaecal Pro kit into the tube and add 800 µL of CD1.
For an extraction negative control, carry out the extraction protocol starting with just the CD1 and no sample.
A positive control should also be extracted if a stock of Positive DNA has not been made previously.
Not to be confused with the Sample Processing Control (SPC) added in Step 2.1; this should be an isolate of an S. Typhi Ty21a, ATCC/NCTC strain, or a confirmed wild type isolate.
Carry out the DNA extraction according to the manufacturer's protocol provided with the DNA extraction kit.
Note that x g and rcf are the same and may be named either way depending on your centrifuge.
In brief:
To the previously added 800 µL of Solution CD1 to the PowerBead tube and 1 µL of validated SPC dilution from the Extraction and Inhibition control kit.
1) Before first use of the SPC, prepare a 1/10th and a 1/100th dilution of the control DNA in pure water and store them on ice.
2) Evaluate the non-diluted and both diluted control DNA solutions in separate extractions by adding 1 µL of control DNA into your reference sample before parallel extractions. Perform the extraction and qPCR and select the dilution factor generating Ct values between 30 and 33.
Homogenize using a vortex adapter at maximum speed for up to 00:10:00 minutes
Centrifuge the PowerBead tube at 15000 x g, Room temperature, 00:01:00
Transfer the supernatant to a clean Microcentrifuge tube, add 200 µL of Solution CD2 then vortex for 00:00:05 seconds
Centrifuge at 15000 x g, Room temperature, 00:01:00 and transfer up to 700 µL of supernatant to a clean 2 mL Microcentrifuge tube, avoiding the pellet
Add 600 µL of Solution CD3 and vortex for 00:00:05 seconds
Load 650 µL onto an MB Spin Column and centrifuge at 15000 x g, Room temperature, 00:01:00
Discard the flow-through and repeat until all the lysate has been passed through the spin column
Carefully place the spin column into a clean 2 mL collection tube. Avoid splashing any flow-through onto the column
Add 500 µL of Solution EA to the spin column and centrifuge at 15000 x g, Room temperature, 00:01:00
Discard the flow-through and add 500 µL of Solution C5 to the spin column and centrifuge at 15000 x g, Room temperature, 00:01:00
Discard the flow through and place the column in a clean 2 mL collection tube
Centrifuge at 16000 x g, Room temperature, 00:02:00 then place the column in a clean 1.5 mL collection tube
Add 50 µL of Solution C6 to the center of the filter membrane and let it sit for 00:01:00 minute
Centrifuge at 15000 x g, Room temperature, 00:01:00 and discard the spin column
The DNA is now ready for qPCR, or storage at -20 °C
Preparation of Primers and Probes
For this assay, six qPCR primer/probe targets are utilised.
For S. Typhi, primers and probes targeting the staG and tviB genes and an intergenic region, currently named C6, are used.
The control triplex targets ttr, a pan-salmonella gene; HF183, a Bacteroides dorei 16s human faecal indicator and the Sample Processing Control (SPC) from Eurogentec.
Before ordering probes, please make sure you select the best fluorophores for the channels of your particular model of qPCR machine you will be using (see guidance note).
| A | B | |
| Primer/Probe Name | Sequence (5' -3') | |
| staG_F | CGC GAA GTC AGA GTC GAC ATA G | |
| staG_R | AAG ACC TCA ACG CCG ATC AC | |
| staG_Pr | [Fluorophore 1] - CA TTT GTT CTG GAG CAG GCT GAC GG - [Quencher] | |
| tviB_F | TGT GGT AAA GGA ACT CGG TAA A | |
| tviB_R | GAC TTC CGA TAC CGG GAT AAT G | |
| tviB_Pr | [Fluorophore 2] - TG GAT GCC GAA GAG GTA AGA CGA GA - [Quencher] | |
| C6_F | GCG ATG TAA TTT ATC TCA CGG AAA | |
| C6_R | AAG TGA GAT TCA TCG CGA TAT TTG | |
| C6_Pr | [Fluorophore 3] - TGG CAT CCC GCG AGA AAG CC - [Quencher] |
Table 1: Primer (F or R) and probe (P) sequences for the S. Typhi Triplex
| A | B | |
| ttr_F | CTC ACC AGG AGA TTA CAA CAT GG | |
| ttr_R | AGC TCA GAC CAA AAG TGA CCA TC | |
| ttr_Pr | [Fluorophore 1] - CA CCG ACG GCG AGA CCG ACT TT - [Quencher] | |
| HF183_F | ATC ATG AGT TCA CAT GTC CG | |
| HF183_R | CTT CCT CTC AGA ACC CCT ATC C | |
| HF183_Pr | [Fluorophore 2] - CT AAT GGA ACG CAT CCC - [Quencher | |
| SPC | [Fluorophore 3] - N/A - [Quencher] |
Table 2: Primer (F or R) and probe (P) sequences for the Assay Control Triplex
Guidance Note:
SPC is sold with either Yakima Yellow or Cy5 fluorophores.
For the Applied Biosystems QuantStudio series of machines, in particular the QS7* Flex, we recommend the following combination of fluorophores and Quenchers for the probes:
| A | B | C | |
| Dye | Quencher | ||
| Fluorophore 1 | FAM | BHQ 1 or QSY | |
| Fluorophore 2 | VIC | BHQ 1 or QSY | |
| Fluorophore 3 | Cy5 | BHQ 3 or QSY 2 |
Table 3: Suggested Fluorophore Dye and Quencher combinations for Tables 1 and 2
*Thermofisher also provide a multiplex option for QuantStudio systems using: FAM, VIC, ABY, JUN, Cy5 and Cy5.5 with their QSY Quenchers, however JUN is not compatible with the ROX in the Takyon Mastermix, and Passive Reference Dye would need to be set to none
Resuspend the probes and primers in the appropriate volume of nuclease free water and make stock dilutions of each primer at 20 micromolar (µM) and each probe at 5 micromolar (µM) .
Preparation of Standards
Using gBlocks gene fragments (IDT) with the target amplicon sequences, a standard curve can be obtained by running a qPCR on a dilution series of the DNA fragments.
This allows determination of potential Ct cut off values to help with determining a positive result in the qPCR with real samples.
A separate protocol for carrying out this experiment is provided in the Typhoid ES workspace.
Quantitative PCR
Thaw qPCR reagents and samples on ice and briefly spin down.
Set up a master mix for the number of samples to be tested plus a positive control, extraction control and qPCR negative control and one extra to allow for pipetting error.
If you wish to carry out single-plex reactions for any of the gene targets, input the same amount of the mastermix, target primer and probe, and substitute the remaining volume with nuclease free water.
| A | B | C | |
| Reagent | Volume per reaction (uL) | Final Concentration (uM) | |
| tviB_F | 0.5 | 0.4 | |
| tviB_R | 0.5 | 0.4 | |
| tviB_Pr | 1 | 0.2 | |
| staG_F | 0.5 | 0.4 | |
| staG_R | 0.5 | 0.4 | |
| staG_Pr | 1 | 0.2 | |
| C6_F | 0.5 | 0.4 | |
| C6_R | 0.5 | 0.4 | |
| C6_Pr | 1 | 0.2 | |
| 2x MasterMix with ROX | 12.5 | 1x | |
| Nuclease free water | 1.5 | - |
Table 4: Mastermix composition for the S. Typhi qPCR
Make up another master mix for the Control Triplex
| A | B | C | |
| Reagent | Volume per reaction (uL) | Final Concentration (uM) | |
| ttr_F | 0.5 | 0.4 | |
| ttr_R | 0.5 | 0.4 | |
| ttr_Pr | 1 | 0.2 | |
| HF183_F | 0.5 | 0.4 | |
| HF183_R | 0.5 | 0.4 | |
| HF183_Pr | 1 | 0.2 | |
| 2x MasterMix with ROX | 12.5 | 1x | |
| 10x Control mix (Eurogentec) | 2.5 | 1x | |
| Nuclease free water | 1 | - |
Table 5: Mastermix composition for the HF183 and extraction control qPCR
Aliquot 20 µL of mastermix to each required well in a 96-well plate.
Add 5 µL of sample, positive control or extraction control; or 5 µL of nuclease free water for negative controls.
Figure 1: Example of a plate map for the qPCR split between both triplexes in use. Positive S. Typhi control, Extraction control (including SPC) and qPCR Negative control are all present.
Seal the plate carefully with a plate seal the spin the plate down briefly to gather all reagents at the bottom of the well and remove bubbles.
Load the plate into the real-time PCR machine after setting it up appropriately and carry out cycling using the following conditions:
| A | B | C | D | |
| Cycle | Temperature (ºC) | Duration | Step | |
| 1 | 50 | 2 minutes | Carry-over prevention | |
| 1 | 95 | 2 minutes | Takyon® activation | |
| 40 | 95 | 15 seconds | Denaturation | |
| 60 | 30 seconds | Annealing | ||
| 72 | 30 seconds | Extension |
Table 6: Cycling conditions for the qPCR. If not automatically set by the software or hardware, the data collection function should be set at the Extension step.
Once the run is complete assess the normalised Ct values for each gene target for each sample, determining positivity using the cut-off Ct values chosen from running the standards.
First, screen the controls in both halves of the Assay to ensure the quality control passes:
1) The positive control should amplify ttr, tviB, staG and C6 - if any of these have not amplified, then there may be an issue with qPCR set up, please refer to the QA/QC Guidance document.
2) The Extraction NTC should have no amplification for all targets except for the SPC - ensure this spike in control is at the appropriate Ct value, as deviance from this may indicate inhibition or loss of amplification efficiency. Amplification of any other target may indicate cross-contamination or non-target amplification. Please refer to the QA/QC Guidance document.
3) qPCR Negative should have no amplification for all targets, including the SPC. Any deviation from this may indicate cross-contamination or non-target amplification. Please refer to the QA/QC Guidance document.
If all gene targets have Ct values higher than the cut-off, check the Ct values for the HF183 target and the spike-in control.
- If the spike-in control is negative or lower than expected this may indicate an issue with PCR inhibition or during DNA extraction
- If the HF183 is negative and spike-in control was positive, then this may mean that there was no or insufficient faecal contamination in the sample site
If all three targets, staG, tviB and C6, are positive, then the sample is considered positive for S. Typhi. If only staG and tviB, or C6 are positive, then it may be suspected positive for S. Typhi and progress to Amplicon Sequencing.
If the sample is positive for ttr and only one of either staG or tviB, repeat the target that was negative in a monoplex reaction as this may result in a positive if the target is just very close to the limit of detection.
The following table gives examples for interpretation of the qPCR results (after monoplex repeats):
| A | B | C | |
| qPCR result | Low Ct values | High Ct values | |
| ttr+ staG+ tviB+ C6+ | S. Typhi | S. Typhi (inhibitors or low concentration) | |
| ttr+ staG+/- tviB+/- C6+ | Suspect S. Typhi | Mixture with low concentration or NTS | |
| ttr+ staG+ tviB+ C6- | Suspect S. Typhi | Mixture with low concentration or NTS | |
| ttr+ staG- tviB+ C6- | Mixture (NTS) | Mixture with low concentration or NTS | |
| ttr+ staG+ tviB- C6- | Mixture (NTS) | Mixture or S. Typhi where Vi gene is lost | |
| ttr+ staG- tviB- C6- | NTS | NTS |
Table 7: Result interpretation