An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

1. Preparation of PCR Template

Note: In order to minimize PCR carryover contamination, it is important to keep several important key steps in the PCR physically-separate from one another: 1) template (DNA) preparation, 2) PCR setup, and 3) gel electrophoresis. It is also recommended to use barrier tips in order to prevent culture or reagent contamination of pipetters.

1. Inoculate 1 ml of Luria Bertani broth or other complex medium (Brain Heart Infusion, Superbroth, etc.) with Salmonella isolate from a single isolated colony. Incubate culture overnight at 37°C.
2. Transfer 1 ml to a sterile, 1.5 ml capacity microfuge tube, centrifuge the bacterial cell suspension at 4,500 xg for ˜ 2 min. to pellet cells.
3. Decant supernatant, resuspend the cell pellet in 1 ml of 100% ethanol and set at room temperature for 10 min. Pellet cells as before (4,500 xg, 2 min.), remove the supernatant and resuspend cells in 1 ml PBS.
4. Centrifuge cells (4,500 xg, 2 min.), discard supernatant and resuspend cells in 1 ml of sterile dH₂O.
5. Dilute the final cell suspension 1/20 in dH₂O (5 μl/95 μl dH₂O) and store at -20°C. The shelf-life of the whole cells as PCR template at -20°C is about one month.

2. PCR Setup

Note: Preparation and dispensing of the PCR reaction mix (template, buffers, oligonucletoideprimers, nucleotides and Taq DNA polymerase) needs to be performed in a physically separate room dedicated and preferably done in a PCR/UV workstation.

Note: The PCR set up room must also be self-contained with a designated -20°C freezer for storage of PCR reagents (buffers, oligonucleotide primers, and nucleotides), enzymes and template, dedicated pipetter set for PCR setup, disposable latex gloves, laboratory coat, barrier tips, microtiter plates, glass capillaries, microfuge tubes, and 10% bleach for decontaminating surfaces. Use a dedicated pipetter set (P10, P100, and P1000) to dispense PCR reagents. This pipette set must never leave the set-up workstation.

Note: Obtain molecular biology or PCR grade dH₂O and aliquot 1 ml into 1.5 ml capacity centrifuge tubes. Dispense aliquoted dH₂O after each use to avoid possible cross-contamination. A similar approach is recommended with the other PCR reagents.

Note: Decontaminate the work area before use with UV illumination and/or wiping down surfaces with 10% bleach. Wear laboratory coat and latex gloves in performing PCR setup. Minimize back and forth traffic from PCR set-up to areas where the PCR reactions are incubated in the thermocylcer and PCR products (amplicons) are separated on agarose gels. If you go back into the PCR set-up area, dispose of the pair of latex gloves you are currently wearing and put on a new pair of gloves.

1. Make a master primer stock in dH₂o to a concentration of 5×10^-4 M. Dilute the primers 1/20 in dH₂O (5 μl/95 μl dH₂o; 25 μM). The oligonucleotide sequence for each flagellin typing primer is described in Table 1.
2. Retrieve PCR reagents and template from -20°C freezer, and allow reagents and samples to thaw out on ice. Leave the Taq DNA polymerase in freezer until needed.
3. Dispense reagents for allelotyping PCR reaction mix as described in Table 1.
4. Dispense 9μl of the PCR reaction mix into each of 10 round bottom wells in a sterile, 96-well, polystyrene microtiter plate, starting at the top of the column (e.g. A1) and moving down the column to the top of the next.
5. Add 1 μl of dH₂O to the 9 μl of PCR mix (no DNA control) to the first well (A1),0.5μl of the positive control templates (i/g,m typing primers- Typhimurium and Enteritidis; r/z10 typing primers- Heidelberg and Hadar; and 1,2/e,n,x typing primers- Typhimurium and Hadar) to 9 μl of the PCR mix in the 2^nd well (B1), and 1 μl of Escherichia coli K12 DH5α to the 9 ml of PCR mix in the third well (C1).
6. For each additional well (D1-H1; A2, B2), add 1 μl of the thawed sample template to the 9 μl of the PCR reaction mix. For the i/g, m allelotyping PCR, S. enterica serovar Typhimurium (i) and Enteritidis (g,m) serve as positive controls, and Salmonella serovars Heidelberg (r) and Hadar (z10) are the positive controls for the r/z₁₀ allelotyping multiplex PCR.
7. Place 10 μl capacity glass capillaries into designated holders for the Idaho Technology Rapidcycler (8).
8. once secured in the holder, load each glass capillary with the PCR reaction mix by touching the capillary to the bottom wells of the microtiter plate. Tilt the holder to allow the liquid to move down the tube and provide space between the ends and the PCR mix before heat sealing the glass tubes with a butane torch to seal both ends of the capillaries.
9. Place the capillary tubes and holder into the Idaho Technology Rapidcycler and use thermocycler programs described in Table 1 for the different allelotyping primers to run the PCR.
10. Proceed to gel electrophoresis step when the thermocycler program is completed.

3. Gel Electrophoresis

Note: Wear a different lab coat designated for use in electrophoresis lab and a new pair of disposable latex gloves.

Note: Wear UV protective eye goggles or face shield and always were gloves when handling ethidium bromide, especially agarose gels.

1. Prepare 100 ml molten 1.5% (w/v) molecular biology grade agarose in 1xTAE (or 1X TBE) electrophoresis buffer (7) by repeatedly heating the agarose suspension in a microwave set at 20% power for 2-5 min intervals with periodic visual inspection. Set the molten agarose in 60°C water bath until it equilibrates to the temperature of the water bath.
2. Set up the gel mold with comb before pouring the molten agarose. Choose a gel comb with sufficient teeth to produce enough wells for controls, samples, and at least 3 molecular weight standards for their placements at the ends and the middle of the agarose gel.
3. Add 2 μl of ethidium bromide (10 mg/ml) to 100 ml of molten 1.5 % (w/v) agarose in 1xTAE (or TBE), gently swirl bottle to mix, avoid producing bubbles, and gently pour the contents of the bottle into the middle of the gel mold for 15 by 10 cm agarose gel. Use the edge of tissue paper or paper towel to remove any bubbles in the agarose gel.
4. Let the gel mold set at room temperature until agarose solidifies (˜30-45 min). Transfer the gel tray with gel & comb to submersible, horizontal electrophoresis chamber containing 1X TAE (or 1X TBE) electrophoresis buffer. Gently remove the comb from the agarose gel.
5. Check placement of the gel tray relative to the cathode or positive pole of the electrophoresis chamber to be sure this pole is at the bottom of the gel. Note: Remember the negatively charged DNA migrates towards the cathode (i.e., “run to red”).
6. Premix 200 μl of DNA molecular weight markers (0.25 μg/μl) with 40 μl of 6X DNA Loading Dye. Load 4.8 μl of the premixed DNA Molecular Weight Marker XIV to the first, middle and last wells.
7. Load each of the PCR samples starting with well 2 and advancing to each subsequent well, moving from left to right. Avoid loading the sample in any of the wells containing the molecular weight standard.
8. To load samples contained in each glass capillary:
   1. Remove each capillary from its holder and etch both ends of the capillary with a glass cutter.
   2. Place thumb and forefinger of both hands on either side of the capillary marked by the glass cutter and snap the capillary.
   3. Place the capillary dispenser on the end opposite of the PCR reaction mix and slowly turn the plunger clockwise until the liquid is at the very bottom of the tube.
   4. Place the capillary into the well to be loaded and slowly turn the plunger clockwise to dispense the Ficoll-weighted sample into the agarose well. Be careful not to puncture the well with the capillary or introduce an air bubble into the well by turning too much past when the last of the liquid leaves the capillary.
9. Once all the samples and standards are loaded, set the constant voltage of the power supply to 90 V (9 volts/cm agarose gel).
10. Discontinue electrophoresis once the yellow dye (Taurazine) front is 1 cm from the bottom of the gel.
11. Once electrophoresis is completed, transfer the gel to a UV transilluminator to visualize DNA fragments and molecular weight standard. Capture image on Polaroid film using a Polaroid camera with orange glass filter (settings: 2 x, and 4.5 y) or as a digital image using a CCD camera and print image with a thermal printer.
12. Place the capillary holders in 10% bleach for 15-30 min. Rinse 3 times with dH₂O and place back in PCR setup room to air dry.

4. Representative Multiplex PCR Results

Results for allelotyping PCR of Salmonella isolates 1-10 are shown in Figure 1 (lanes 3-12) and interpreted as follows. An O allele designation is given to Salmonella isolate based on amplicon (PCR product) matching in size to the PCR control and as predicted for the O allele primer sets used (3): B- 560bp, C1- 340bp, C2- 400 bp, D1- 620 bp, and E1- 280 bp. For isolates tested with the O allelotyping PCR (Fig. 1A), we assigned O alleles B (lanes 10-13), C1 (lanes 7-9), C2 (lanes 4, 5), D1 (lane 3), and E1 (lane 6) to the ten Salmonella isolates tested in lanes 3-12. These same Salmonella isolates were tested, in the same order as Fig. 1A, for H1 alleles i; g,m; r; and z10 (Fig. 1B,C). Again, a H1 allele is assigned to each isolate dependent on the presence of an amplicon matching in size to the PCR control and as predicted for the 4 H1 allele primer sets used (3): i- 510 bp (Fig. 1B, lane 2); g,m- 310 bp (Fig. 1B, lane 2); r- 170 bp (Fig. 1C, lane 2); and z10- 360 bp (Fig. 1C, lane 2). H1 alleles were assigned to one of the ten Salmonella isolates: i – isolates 3 and 8 (Fig. 1B, lanes 5 & 10); g,m- isolates 1 and 5 (Fig. 1B, lanes 3 & 7), r to isolates 6 and 9 (Fig. 1C, lanes 8 & 11); and z10 to isolates 2, 7, and 10 (Fig. 1C, lanes 4, 9, &12). Salmonella isolate 4 was negative for the four H1 alleles tested. Finally, Salmonella isolates were tested by PCR for H2 alleles associated with the 1,2; 1,5; 1,6; 1,7 antigen complex and e,n,x; e,n,z15 antigen complex. The primer sets for the H2 1,2; 1,5; 1,6; 1,7 complex and H2 e,n,x; e,n,z15 complex produce 290 bp and 150 bp amplicons, respectively (3). The primer sets cannot distinguish specific H2 alleles within either H2 antigen complex to assign a definitive allele type, ex. 1,2, to an isolate (3). Salmonella isolates 4, 6, 8-10 were positive for H2 alleles associated with H2 1,2; 1,5; 1,6; 1,7 antigen complex, while isolates 2 and 7 were positive for H2 alleles associated with e,n,x; e,n,z15 antigen complex (data not shown). While Salmonella isolates 1, 3, and 5 were negative for the two H2 antigen complexes, only isolates 1 and 5 were negative for H2 flagellin, as determined using a generic H2 flagellin PCR (1) (data not show). These results are summarized in Table 2 along with the presumptive Salmonella serovar assigned to each isolate.

Figure 1. Multiplex PCR for Identifying Specific O and H Alleles Associated with S. enterica Serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. (A) O Allele Multiplex PCR. (B,C) H1 Allele Multiplex PCR for Identifying Flagellin Alleles: i/g,m (B) and r/z10 (C). Lanes 1and 13: 100 bp ladder (Promega; Madison, WI); lane 2: multiplex PCR controls for O alleles B, C1, C2, D1, and E (A), H1 alleles i/g,m (B), and H1 alleles r/z10 (C); and lanes 3-12: Salmonella isolates 1-10 (Table 2).

Table 1. Protocol for Salmonella flagellinallelotyping PCR: composition, conditions, and expected results.

*The working stock concentration of PCR oligonucleotide primers is 25 μM

Table 2. Allelotyping PCR Results (Fig. 1) and Salmonella Serovar Designation Based on Identification of O, H1, and H2 Alleles

> ¹H2 PCR produces a 1.5 kb amplicon for all Salmonella possessing H2 flagellin, regardless of H2 allele (1). This PCR is used to distinguish monophasic from the biphasic Salmonella.
²H2 multiplex PCR cannot distinguish among the different alleles for the H2 1,2; 1,5; 1,6; 1,7 antigen complex (3).
³Phage conversion of S. enterica serovar Hadar alters LPS O antigen to produce serovar Istanbul. The O allelotyping PCR cannot discern these subtle genetic/antigenic changes.

Table 3.Salmonella enterica Serovars Identified by Allelotyping PCR

¹The serovars highlighted in bold are the ones more commonly encountered by diagnostic or food microbiology laboratory.
²H2 PCR produces a 1.5 kb amplicon for all Salmonella possessing H2 flagellin, regardless of H2 allele (1). This PCR is used to distinguish monophasic from the biphasic Salmonella.
³Phage conversion of S. enterica serovar Hadar alters LPS O antigen to produce serovar Istanbul. The O allelotyping PCR cannot discern these subtle genetic/antigenic changes.