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

[DESCRIPTION]
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.

[STEPS]
SECTION: Bacterial Lysis (BAX lysis) Procedure to prepare DNA template
2.1. Reaction: 
Add 100 µL Protease/Lysis Buffer per well in the PCR plate
Add 2.5 µL Sample (overnight culture) per well
SECTION: Bacterial Lysis (BAX lysis) Procedure to prepare DNA template
2. 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). 
SECTION: Bacterial Lysis (BAX lysis) Procedure to prepare DNA template
3. Move on to PCR or freeze lysates
SECTION: Bacterial Lysis (BAX lysis) Procedure to prepare DNA template
1. Prepare the BAX Lysis Buffer: Add 150ul Protease solution into 12mL Lysis Buffer. 

NOTE: this solution is stable at 4C for two weeks
SECTION: Bacterial Lysis (BAX lysis) Procedure to prepare DNA template
2.2. 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
SECTION: HPS Multiplex PCR Reaction
4. Make up HPS master mix following Table 2. Store at -20 °C.
SECTION: HPS Multiplex PCR Reaction
5. Thaw master mix, vortex, aliquot 20 µL per well in PCR plate.
SECTION: HPS Multiplex PCR Reaction
6. Add 5 µL of lysate (if frozen, thaw and gently pipette to resuspend) per well.  Final reaction volume is 25 µL.

SECTION: HPS Multiplex PCR Reaction
7. Seal plate with Microseal B and place in thermocycler.

SECTION: HPS Multiplex PCR Reaction
8. Run program according to Table 3.


SECTION: HPS Multiplex PCR Reaction
9. Once complete, begin gel or store PCR products at -20 degrees C.
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
10. Make a 2% agarose gel (in 0.5L flask, dissolve with heat 5g agarose in 250mL of 1X TBE) and cast in 15x25cm array.
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
11. After gel is setup, transfer to buffer tank containing enough 1X TBE to cover gel and remove combs.    
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
12. 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.  
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
13. Flank with control strain S. Typhimurium ATCC – 14028S (7) on either side of reaction wells.
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
14. Run gel at 170 volts for approximately 60 minutes.
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
15. Staining
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
15.1. 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)
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
16. Image with a UV imager (Figure 1). 

SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
15.2. 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.
SECTION: Agarose gel electrophoresis and staining, to visualize DNA amplicons
15.3. 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.