Protocol for Identifying Highly Pathogenic Salmonella Using the HPS Multiplex PCR Assay
Gregory P Harhay, Dayna Harhay, Kerry D. Brader, Jim Bono, Mick Bosilevac, Tatum S Katz
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
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.
Attachments
Steps
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).
