Oct 03, 2024
Nested PCR amplification of Salmonella Typhi from extracted wastewater concentrate
- Shannon Fitz1,
- Alex Shaw1,
- michael Owusu2,
- Dilip Abraham3,
- Anton Spadar4
- 1Imperial College London;
- 2Kwame Nkrumah University of Science and Technology;
- 3CMC Vellore;
- 4London School of Hygiene and Tropical Medicine
- GEMS - Genomic Environmental Microbial Surveillance
Protocol Citation: Shannon Fitz, Alex Shaw, michael Owusu, Dilip Abraham, Anton Spadar 2024. Nested PCR amplification of Salmonella Typhi from extracted wastewater concentrate. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqnw3ogk5/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: October 03, 2024
Last Modified: October 03, 2024
Protocol Integer ID: 109018
Keywords: PCR, salmonella typhi, nested PCR
Funders Acknowledgement:
The Gates Foundation
Grant ID: INV-049092 PA4273
Abstract
This standard operating procedure describes nested PCR protocols for the generation of amplicons from V. cholerae and S. typhi.
PCR primers have been sourced from the literature or designed using Geneious, with primer pooling tested to minimise primer-primer interactions. Whilst the first round of PCR may be sufficient alone for detection (and will yield larger amplicons) a second round of PCR has been included to increase sensitivity.
Materials
DreamTaq PCR Master Mix (2X)Thermo FisherCatalog #K1071
Cost per sample: £0.90
28 primers required (cost excluded from estimate as primers do not need to be ordered each time)
Dreamtaq cost per sample: ~£0.45 per sample
Extra equipment required:
thermocycler, vortex, mini centrifuge
Primer preparation step
Primer preparation step
| A | B | C | D | |
| Primer name | Primer | Pool G | Pool B | |
| 3.1.1_4.3.1.1.P1_F | CCGGATATCGATCACCCCAAA | G | B | |
| 3.1.1_4.3.1.1.P1_R | GCTTTCGTGATGTCGCTCAG | G | ||
| 3.1.1_Inner_Rev | ACACGGCCACAGTAAGGG | B | ||
| 4.3.1_4.3.1.2_1_F | AGAATTTTCGCATCAGGGGGAT | G | ||
| 4.3.1_4.3.1.2_1_R | CCAGTCATTAACGAGGCGCTT | G | ||
| 4.3.1.2_Inner_For | CGTCGCCTCGGTTTTAACATTA | B | ||
| 4.3.1.2_Inner_Rev | CGCTGATGAATCCGCACTC | B | ||
| tviD_842_F | TGCAAGCTGCTTAGTGATCGA | G | B | |
| tviD_842_R | TGAGTCCGGTAAAACGAGCTC | G | ||
| tviD_Inner_R | CAGGATGGATATTCCCTGCGT | B | ||
| 1_3_4.3.1_F | ACGATGGTACTGAACAACCCT | G | ||
| 1_3_4.3.1_R | TACGCTGTTCAGCCCGATATC | G | ||
| 2.2.2_2_F | AGCACAGTTCATCCGAGTGAT | G | ||
| 2.2.2_2_R | AGCATCAGACTCTGCGACAC | G | ||
| 2.5_4.3.1.2.1_3.3_F | CGGTTCGTTGTCCATTTCGG | G | ||
| 2.5_4.3.1.2.1_3.3_R | GGCGGCTTTCTTCAGTTTTTCA | G | ||
| 3.3.1_3.3_F | GCGAAATCGTTCCCGGAAAAA | G | ||
| 3.3.1_3.3_R | TCCATCGGAAAGCCTTCGTAA | G | ||
| 4.3.1.1_F | TCTGGCCTGATACCTGGATGT | G | ||
| 4.3.1.1_R | CGATCGGATATCCAGCACCA | G | ||
| gyrA_F | TGACGCCTTCTTCGTACTCAC | G | ||
| gyrA_R | CTGAAGCTGATCGCCGATAAAC | G | ||
| parC_F | GCCAGACGACCGAACATATGT | G | ||
| parC_R | TTCCTCGCAACCTGTCTCAC | G | ||
| 4.3.1.2.1.1_4_F | GTCAGGCCTGGTTTGACAATC | G | ||
| 4.3.1.2.1.1_4_R | CCTGTGAACTAACCCCTGCA | G | ||
| 2.3.2_F | GACGATAAACCGCTTCCGTCA | G | ||
| 2.3.2_R | AGCCGGGTACAGTAGTCCAA | G |
Primer preparation
- Reconstitute primers to 100 µM using nuclease-free water
- Create working stocks of 10 µM using nuclease-free water
- Create 10 µM primer pools G and B by mixing together 10 µL of each primer marked as being part of the pool. Scale up as needed.
Workstation Preparation
Clean the PCR areas before setting up the reactions; clean room for the master mix and an area for the addition of the template.
- Clean the working area and pipettors with an approved DNAse inhibitor solution.
- Clean the Class II biosafety cabinet and pipettes with 70% ethanol and wipe with a paper towel.
Place a disposal Dispo-safe “sweetie” jar or bio-bin on the bench.
Sample Retrieval
Place a rack inside a polystyrene sample box for sample transfer.
Remove tube containing the required samples from the -80 °C freezer.
Close freezer and polystyrene box.
Allow the samples to thaw on ice.
Round 1 PCR reaction
Prepare the following Master mix in a 1.5ml Eppendorf Lobind tube:
| 1 Reaction (µL) | Reactions | ||
| DreamTaq 2x master mix | 12.5 | µL | |
| Water | 6.5 | µL | |
| Primer pool G | 1 | µL | |
| Total volume | 20 |
Briefly vortex and centrifuge down the master mix and aliquot 20 µL into each PCR tube.
Add 5 µL of extracted DNA from each sample to a tube, mix well by pipette and label accordingly.
Briefly centrifuge down the PCR mixes and store on ice.
Amplify using the following cycling conditions:
| A | B | C | D | |
| CYCLE | STEP | TEMP (°C) | TIME | |
| 1 | Initial Denaturation | 95 | 2 minutes | |
| 35 | Denaturation | 95 | 30 seconds | |
| Annealing | 56 | 30 seconds | ||
| Extension | 72 | 3 minutes | ||
| 1 | Final Extension | 72 | 10 minutes | |
| - | Hold | 10 | - |
Round 2 PCR reaction
Prepare the following Master mix in a 1.5ml Eppendorf Lobind tube:
| 1 Reaction (µL) | Reactions | ||
| DreamTaq 2x master mix | 12.5 | µL | |
| Water | 6.5 | µL | |
| Primer pool B | 1 | µL | |
| Total volume | 20 |
Briefly vortex and centrifuge down the master mix and aliquot 20 µL into each PCR tube.
Add 5 µL of extracted DNA from each sample to a tube, mix well by pipette and label accordingly.
Briefly centrifuge down the PCR mixes and store on ice.
Amplify using the following cycling conditions:
| A | B | C | D | |
| CYCLE | STEP | TEMP (°C) | TIME | |
| 1 | Initial Denaturation | 95 | 2 minutes | |
| 35 | Denaturation | 95 | 30 seconds | |
| Annealing | 55 | 30 seconds | ||
| Extension | 72 | 1 minute | ||
| 1 | Final Extension | 72 | 10 minutes | |
| - | Hold | 10 | - |
Gel electrophoresis bands
| First round amplicon length (bp) | Second round amplicon length (bp) | ||
| 3.1.1. | 2991 | 263 | |
| 4.3.1.2 | 2725 | 398 | |
| tviD | 843 | 238 |
Protocol references
All primers were designed by Dr Anton Spadar, unpublished.