May 02, 2024
Protocol for Identifying Highly Pathogenic Salmonella Using the HPS Multiplex PCR Assay
- Dayna Harhay1,
- Kerry D. Brader1,
- Jim Bono1,
- Gregory Harhay1,
- Mick Bosilevac1,
- Tatum Katz1
- 1US Meat Animal Research Center, Agricultural Research Service, US Department of Agriculture, Clay Center, NE, USA
Protocol Citation: Dayna Harhay, Kerry D. Brader, Jim Bono, Gregory Harhay, Mick Bosilevac, Tatum Katz 2024. Protocol for Identifying Highly Pathogenic Salmonella Using the HPS Multiplex PCR Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr3on3vmk/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 06, 2023
Last Modified: May 02, 2024
Protocol Integer ID: 84558
Keywords: Non-typhoidal Salmonella enterica, NTSE, Highly Pathogenic Salmonella, HPS, Polymerase Chain Reaction, PCR, multiplex PCR assay, virulence genes, pathogenicity, NP108, CRIS 3040-42000-020-00D, Meat Safety and Quality Research Unit, salmonella serotype, highly pathogenic salmonella, pathogenic salmonella, salmonella isolate, complete closed salmonella genome sequence, salmonella sample, salmonella, salmonellosi, most cases of salmonellosi, hps multiplex pcr assay, multiplex pcr assay for the identification, hps gene target, hps multiplex pcr assay this protocol, multiplex pcr assay, indication of the potential pathogenicity, virulence gene, top 20 serotype, potential pathogenicity, particular serotype, comparative genomic research, different serotype, serotype, validation of this assay, hps index, gene, genome, identifying hp, absent among serotype, recent addition to the amrfinderplus reference gene catalog, utility of this assay
Funders Acknowledgements:
CRIS
Grant ID: 3040-42000-020-00D
Disclaimer
The use of product and company names is necessary to accurately report the methods and results; however, the United States Department of Agriculture (USDA) neither guarantees nor warrants the standard of the products, and the use of names by the USDA implies no approval of the product to the exclusion of others that may also be suitable. The USDA is an equal opportunity provider and employer.
Abstract
This protocol describes how to perform a multiplex PCR assay for the identification of Highly Pathogenic Salmonella (HPS). The assay targets eight virulence genes including: sseK2 (HPS-6), sseK3 (HPS-1), avrA (HPS-3), lpfB (HPS-5), spvD (HPS-4), sspH2 (HPS-7), gtgA (HPS-2) and invA (i). Comparative genomic research using 23 complete closed Salmonella genome sequences (1-23) revealed these genes to be shared among Salmonella serotypes noted for being invasive and/or causing most cases of salmonellosis (i.e. consistently on the CDC’s list of top 20 serotypes attributed to human illness) but to varying degrees are absent among serotypes that are less frequently associated with human illness. Validation of this assay with 1303 Salmonella of 69 different serotypes confirmed the utility of this assay for identifying HPS (Harhay et. al., in preparation). Salmonella samples that result in the amplification of five or more targets are identified as HPS, and the markers amplified are reported as an HPS index (HPSi) (Figure 1). The assay described is intended for use with DNA lysates of Salmonella isolates, not lysates of enrichment cultures, where more than one Salmonella serotype may be present. Notably, the HPS assay is not serotype specific (i.e. the results do not indicate the presence of a particular serotype) rather it provides an indication of the potential pathogenicity of the Salmonella being assayed. This protocol includes information on generation of template DNA, primer sequences needed (Table 1), construction of the PCR master mix (Table 2), thermal cycler program used for amplification (Table 3), parameters for DNA gel electrophoresis, and amplicon visualization and scoring (Figure 1). Further evidence of the utility of the HPS gene targets is their recent addition to the AMRFinderPlus Reference Gene Catalog.
Materials
Bacterial Lysis (BAX lysis) Procedure to prepare DNA template
Equipment:
Thermal cycler (BIO-RAD T100)
Lysis Program (see below)
PCR plates & Microseal B (BIO-RAD; #MSB-1001)
Multi-Channel Pipettors
Buffers and reagents:
BAX Lysis Buffer (Hygiena; item #ASY2011; ref. #D14403062)
BAX Protease Vial (Hygiena; item #ASY2012; ref. #D11134081)
Control strain (S. Typhimurium ATCC - 14028S (7))
Overnight Culture of samples in question
HPS Multiplex PCR Reaction
Equipment:
Thermal cycler (BIO-RAD T100)
Program (Table 3)
PCR plates & Microseal B (BIO-RAD; #MSB-1001)
Multi-Channel Pipettors (10ul and 20ul 12 channel preferrable)
Buffers and reagents:
Bullseye HS Taq Pol (MidSci; BE225108)
Deoxynucleotide (dNTP) Solution Mix (New England Biolabs; N0447L)
Primer list: Table 1.
Master Mix: Table 2.
Agarose gel electrophoresis and staining, to visualize DNA amplicons (see Figure 1)
Equipment:
4x26-Well Combs, Fixed Height, 1.5 mm Thickness, Multichannel Pipet Compatible (BIO-RAD; #1704457)
Sub-Cell GT Horizontal Electrophoresis System, 15 x 25 cm tray, with gel caster (BIO-RAD; #1704484)
0.5 L Erlenmeyer flask
Ethidium Bromide Extractor (GVS; item #10448031). Use as per manufacturers instruction.
Buffers and reagents:
HPS Marker generated using Control strain (S. Typhimurium ATCC - 14028S (7))
10x TBE (Fisher Scientific; #BP1333) - NOTE: it is important to use TBE; buffers such as SB result in poor band separation.
Bullseye General Purpose Agarose GP2 (MidSci; #BE-A500)
6x loading dye (purchased or homemade)
Tables and Figures
Table 1. HPS primer list
Table 2. HPS Master Mix
Table 3. Thermal cycler program adapted from (24)
Figure 1. Example agarose gel depicting the various HPS banding patterns.
Troubleshooting
Bacterial Lysis (BAX lysis) Procedure to prepare DNA template
Prepare the BAX Lysis Buffer: Add 150ul Protease solution into 12mL Lysis Buffer.
NOTE: this solution is stable at 4C for two weeks
In a PCR plate, dispense 100 µL of the BAX lysis buffer per well, add 2.5 µL of overnight culture, seal with Microseal B, and place in thermal cycler. Run Lysis Protocol listed below (35 minutes).
Reaction:
Add 100 µL Protease/Lysis Buffer per well in the PCR plate
Add 2.5 µL Sample (overnight culture) per well
Lysis Thermal Cycler Program:
37 C – 20 min
95 C – 10 min
Cool 4 C – 5 min
NOTE: make sure lid of thermal cycler is set to 96C
Move on to PCR or freeze lysates
HPS Multiplex PCR Reaction
Make up HPS master mix following Table 2. Store at -20 °C.
Thaw master mix, vortex, aliquot 20 µL per well in PCR plate.
Add 5 µL of lysate (if frozen, thaw and gently pipette to resuspend) per well. Final reaction volume is 25 µL.
Seal plate with Microseal B and place in thermocycler.
Run program according to Table 3.
Once complete, begin gel or store PCR products at -20 degrees C.
Agarose gel electrophoresis and staining, to visualize DNA amplicons
Make a 2% agarose gel (in 0.5L flask, dissolve with heat 5g agarose in 250mL of 1X TBE) and cast in 15x25cm array.
After gel is setup, transfer to buffer tank containing enough 1X TBE to cover gel and remove combs.
To completed HPS PCR reaction plate, gently remove the Microseal B cover tape, add 6 µL of 6X loading dye to the reaction. Pipette up and down to mix, then with same tip, transfer 6 µL of mixture to the gel and dispense in an appropriate well.
Flank with control strain S. Typhimurium ATCC – 14028S (7) on either side of reaction wells.
Run gel at 170 volts for approximately 60 minutes.
Staining
After electrophoresis, stain the gel with Ethidium Bromide (EtBr) (~1 μg/mL: from a 1% stock solution, add 10 μL EtBr per 100 mL dH2O) for 30 min. Use a BioRad staining tray with a lid and place this on a rocker to gently agitate the stain with the gel in the tray.
NOTE: use caution and appropriate PPE with EtBr (Toxic if inhaled; Suspected of causing genetic defects)
Carefully pour off the stain (use gloves and appropriate PPE) into an Ethidium Bromide Extractor (see Materials) or use similar means of safely disposing of the staining solution.
Add 100ml of water to the tray to de-stain the gel for 15-20 minutes. Again, use a rocker on a low setting. Dispose of the de-stain water appropriately.
Image with a UV imager (Figure 1).
Protocol references
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