Jul 10, 2023
Version 3
Extraction and qPCR of Environmental Surveillance samples for the detection of Salmonella Typhi V.3
- Dilip Abraham1,
- Nicola Elviss2,
- Nicholas Feasey3,
- Nick Grassly4,
- Jacob John1,
- Gagandeep Kang1,
- John Scott Meschke5,
- Venkata Raghava Mohan1,
- Satheesh Nair2,
- Jonathan Rigby3,
- Rajan Srinivasan1,
- Catherine Troman4,
- Christopher Uzzell4,
- Nicolette Zhou5
- 1Christian Medical College, Vellore, India;
- 2UK Health Security Agency;
- 3Malawi Liverpool Wellcome Trust Clinical Research Program, Blantyre, Malawi;
- 4Imperial College London;
- 5University of Washington
Protocol Citation: Dilip Abraham, Nicola Elviss, Nicholas Feasey, Nick Grassly, Jacob John, Gagandeep Kang, John Scott Meschke, Venkata Raghava Mohan, Satheesh Nair, Jonathan Rigby, Rajan Srinivasan, Catherine Troman, Christopher Uzzell, Nicolette Zhou 2023. Extraction and qPCR of Environmental Surveillance samples for the detection of Salmonella Typhi. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5zrw4v1b/v3Version created by Catherine Troman
Manuscript citation:
Uzzell C, Troman C, Rigby J, Mohan V, John J, Abraham D, Srinivasan R, Nair S, Meschke J, Elviss N, Kang G, Feasey N, Grassly N (2021) Environmental surveillance for Salmonella Typhi as a tool to estimate the incidence of typhoid fever in low-income populations. medRxiv, doi: 10.1101/2021.05.21.21257547
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: June 05, 2023
Last Modified: April 10, 2024
Protocol Integer ID: 82877
Keywords: Salmonella, Typhi, Paratyphi, wastewater, environmental surveillance
Funders Acknowledgement:
Bill and Melinda Gates Foundation
Disclaimer
Please note, the author list is in alphabetical order and does not reflect contribution.
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
DNA Extraction
DNA Extraction
For extraction from Moore swabs, empty the contents of a PowerBead tube provided in the extraction kit into the PowerBead tube used for storing the filter.
For the pellet from membrane filtration, resuspend the pellet in 800µl CD1 from the first step of the DNA extraction protocol, then transfer to the PowerBead tube for homogenisation.
For an extraction negative control, carry out the extraction protocol starting with just the CD1 (step2.1) and no sample.
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:
Add 800µl of Solution CD1 to the PowerBead tube and 1ul 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 the 2 diluted control DNA solutions in separate extractions by adding 1μL 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 10 minutes
Centrifuge the PowerBead tube at 15000 x g, 00:01:00
1m
Transfer the supernatant to a clean Microcentrifuge tube, add 200µl of Solution CD2 then vortex for 5s
Centrifuge at 15000 x g, 00:01:00 and transfer up to 700µl of supernatant to a clean 2ml Microcentrifuge tube, avoiding the pellet
Add 600µl of Solution CD3 and vortex for 5s
Load 650µl onto an MB Spin Column and centrifuge at 15000 x g, 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 2ml collection tube. Avoid splashing any flow-through onto the column
Add 500µl of Solution EA to the spin column and centrifuge at 15000 x g, 00:01:00
Discard the flow-through and add 500µl of Solution C5 to the spin column and centrifuge at 15000 x g, 00:01:00
Discard the flow through and place the column in a clean 2ml collection tube
Centrifuge at 16000 x g, 00:02:00 then place the column in a clean 1.5ml collection tube
Add 50µl of Solution C6 to the center of the filter membrane and let it sit for 1 minute
Centrifuge at15000 x g, 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
Preparation of Primers and Probes
S. Typhi genes being targeted are staG, tviB, and ttr. Primers and probes have also been included for HF183 human faecal indicator to use as a positive control for faecal contamination in the samples and sample sites.
Before ordering probes, please make sure the selected fluorophores are compatible with the 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_P | [Cy5] - CA TTT GTT CTG GAG CAG GCT GAC GG - [BHQ2] | |
| ttr_F | CTC ACC AGG AGA TTA CAA CAT GG | |
| ttr_R | AGC TCA GAC CAA AAG TGA CCA TC | |
| ttr_P | [FAM] - CA CCG ACG GCG AGA CCG ACT TT - [BHQ1] | |
| tviB_F | TGT GGT AAA GGA ACT CGG TAA A | |
| tviB_R | GAC TTC CGA TAC CGG GAT AAT G | |
| tviB_P | [JOE] - TG GAT GCC GAA GAG GTA AGA CGA GA - [BHQ1] | |
| HF183_F | ATC ATG AGT TCA CAT GTC CG | |
| HF183_R | CTT CCT CTC AGA ACC CCT ATC C | |
| HF183_P | [FAM] - CT AAT GGA ACG CAT CCC - [BHQ1] |
Table1: Primer (F or R) and probe (P) sequences. Fluorescent dyes and quenchers are shown in square brackets.
Resuspend the probes and primers in the appropriate volume of nuclease free water and make stock dilutions of each primer at 20µM and each probe at 5µM.
Preparation of Standards
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.
| A | B | |
| gene target | gBlocks sequence (5' - 3') | |
| staG | CGGCGCGAAGTCAGAGTCGACATAGGCATAGATTTTCAGGCCATACATTAATTTGCCAAGGTTGCTATAAACATTTGTTCTGGAGCAGGCTGACGGAAATTCCGTGAACTCGCTGGTGATCGGCGTTGAGGTCTTATC | |
| ttr | GAAACGCTGAACGGACTCACCAGGAGATTACAACATGGCTAATTTAACCCGTCGTCAGTGGCTAAAAGTCGGTCTCGCCGTCGGTGGGATGGTCACTTTTGGTCTGAGCTACCGTGATGTGGCGA | |
| tviB | CTTGATTTGACTTCCGATACCGGGATAATGCCATACTCTCGTCTTACCTCTTCGGCATCCACCCATGGATCAAAAATATCCACTTTACAACTATATTTACCGAGTTCCTTTACCACATCAATAAT | |
| HF183 | GGGATCATGAGTTCACATGTCCGCATGATTAAAGGTATTTTCCGGTAGACGATGGGGATGCGTTCCATTAGATAGTAGGCGGGGTAACGGCCCACCTAGTCAACGATGGATAGGGGTTCTGAGAGGAAGGTC |
Table 2: Sequences used for gBlocks gene fragments. Sequences for the S.Typhi targets were taken from the CT18 reference genome (AL513382.1) and the HF183 sequence was taken from a Bacteroides dorei 16S rRNA partial gene sequence (MT464394.1). 3 additional bases were added either side of the staG and HF183 amplicon sequences. 15 and 8 bases were added to either end of the ttr and tviB sequences respectively to reach the minimum length for the gBlocks fragment.
Quantitative PCR
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 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) | |
| ttr_F | 0.25 | 0.2 | |
| ttr_R | 0.25 | 0.2 | |
| ttr_P | 0.5 | 0.1 | |
| tviB_F | 0.5 | 0.4 | |
| tviB_R | 0.5 | 0.4 | |
| tviB_P | 1 | 0.2 | |
| staG_F | 0.5 | 0.4 | |
| staG_R | 0.5 | 0.4 | |
| staG_P | 1 | 0.2 | |
| 2x MasterMix with ROX | 12.5 | 1x | |
| Nuclease free water | 2.5 | - |
Table 3: Mastermix composition for the S.Typhi qPCR
Make up another master mix for the HF183 qPCR and internal control
| A | B | C | |
| Reagent | Volume per reaction (uL) | Final Concentration (uM) | |
| HF183_F | 0.5 | 0.4 | |
| HF183_R | 0.5 | 0.4 | |
| HF183_P | 1 | 0.2 | |
| 2x MasterMix with ROX | 12.5 | 1x | |
| 10x Control mix (Eurogentec) | 2.5 | 1x | |
| Nuclease free water | 3 | - |
Table 4: Mastermix composition for the HF183 and extraction control qPCR
Aliquot 20µl of mastermix to each required well in a 96-well plate.
Add 5µl of sample, or 5µl of nuclease free water for negative controls.
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 | |
| Cycle | Temperature (ºC) | Duration | |
| 1 | 50 | 2 minutes | |
| 1 | 95 | 2 minutes | |
| 40 | 95 | 15 seconds | |
| 60 | 30 seconds | ||
| 72 | 30 seconds |
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.
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 are positive, then the sample is considered positive for S.Typhi.
If the sample is positive for ttr and only one of either staG or tviB, repeat the target that was negative in a singleplex reaction as this may result in a positive if the target is just very close to the limit of detection.
The following table gives expamples for interpretation of the qPCR results (after singleplex repeats):
| A | B | C | |
| qPCR result | Low Ct values | High Ct values | |
| ttr+ staG+ tviB+ | S.Typhi | S.Typhi (or a more complex mixture but unlikely) | |
| ttr+ staG- tviB+ | Mixture (NTS) | Mixture (NTS) | |
| ttr+ staG+ tviB- | Mixture (NTS) | Mixture or S.Typhi where Vi gene is lost |