PX (Xbal)



PAH1 (Hhal)



PAT1 (Taql)



IRS-PCR, infrequent restriction site polymerase chain raction.

IRS-PCR, infrequent restriction site polymerase chain raction.

21. pGEM marker.

22. Mini Protean electrophoresis cell and power supply (Bio-Rad).

23. Long-wave UV transilluminator for visualization of polyacrylamide gels (UltraLum).

24. MP4 Land camera (Polaroid).

2.4. Computerized Analysis of Electrophoretical Patterns

1. Scanner ScanJet IICX (Hewlett Packard).

2. Software GelCompar 4.0 or superior (Applied Maths).

3. PC personal computer.

4. Laser printer.

3. Methods

3.1. Pulsed-Field Gel Electrophoresis

1. Grow the isolates on trypticasein soy agar plates overnight at 37°C. Inoculate cultures into 6 mL of nutrient broth and incubate at 37°C with shaking until growth reaches an absorbance of 0.12 at 650 nm (1 x 109 cells/mL).

2. Spin 5 mL of cells in a refrigerated centrifuge (Beckman GS-6R or similar) at 3000g for 10 min and decant supernatant. Wash the cells with 5 mL ice-cold TES buffer and spin once again. Discard supernatant and resuspend pellet in 0.3 mL TES buffer.

3. Prepare by boiling a solution of l% (w/v) low-melt agarose in distilled water and maintain at 56°C in a water bath. Mix 700 ^L of warm agarose with the 300 ^L of bacterial suspension and dispense into a block mold, allowing the agarose blocks to solidify by placing them at 4°C for 15-30 min.

4. Add lysozyme and RNAse to the lysis buffer and incubate the blocks for 2 h at 37° C in 3 mL of this lysis buffer.

5. Discard the lysis buffer and add 3 mL ESP buffer. Add the Proteinase K to ESP buffer just before use in a final concentration of l mg/mL. Incubate overnight at 56°C in a waterbath.

6. Discard the ESP buffer carefully, washing the blocks with 3 mL distilled water for 15 min with gentle agitation at room temperature. Discard the water and add 2 mL TE buffer and 30 ^L of l00 mM PMSF solution for each sample (see Note 2). Caution: This step has to be performed in a fume hood and wearing gloves. Agitate gently at room temperature for 30 min. Repeat this step with fresh TE buffer and PMSF solution.

7. Remove TE buffer and PMSF solution, add 3 mL of distilled water, and incubate samples at room temperature for 15 min with gentle agitation.

8. Remove water, add 3 mL of TE buffer, and incubate at room temperature for 30 min with gentle agitation. Repeat this step twice.

9. Remove the buffer and add 3 mL of new TE buffer. Keep the samples refrigerated.

10. Cut a small piece of the block, place it in an Eppendorf tube with 100 ^L of the restriction enzyme buffer, and incubate at room temperature for 1 h. Remove buffer carefully with a pipet and replace with 20 U of the enzyme, buffer, and distilled water in a final volume of 50 ^L. Incubate at 37°C for 4 h.

11. Prepare 110 mL of 1.2% (w/v) Pulsed-Field Certified Agarose in 0.5X TBE buffer and cast the gel. When gel hardens, cast the gel wells with the agarose blocks, making sure they are placed flat against the leading edge of the well. Insert polymerized phage lambda DNA agarose block as a molecular size marker. Seal the wells with warm agarose.

12. Transfer the gel together with 2 L of cold 0.5X TBE buffer with thiourea to the machine (CHEF-DRII) and leave for 15 min for equilibrium.

13. Program the pulse machine and run the electrophoresis for the established voltages, times, and pulses, with a constant electrophoresis buffer temperature of 12°C (Table 3).

14. Dilute the stock solution of ethidium bromide to reach a concentration of 2 ^g/mL. Incubate the gel in obscurity for 30 min. Wash the gel with distilled water and photograph the gel under 254-nm UV transillumination.

15. Interpret the gel visually or use the computerized analyzer (see Note 5). Tenover et al. (8) describe several criteria for strain typing through the use of PFGE DNA restriction patterns.

3.2. PCR Fingerprinting

1. Grow the isolates overnight at 37°C in trypticasein soy agar plates.

2. Resuspend in an Eppendorf tube a small loopful of bacteria in 500 ^L of sterile water. Boil for 10 min and spin at 11,600g for 10 min. Measure the DNA concentration of the supernatant by spectrophotometry and prepare DNA stock solutions of 50 ng/^L. Keep at -20°C.

3. Carry out a PCR reaction with 10 mMTris-HCl, pH 8.3, 50 mM KCl, 3 mMMgCl2, 200 ^M of each dNTPs, 1 U AmpliTaq polymerase, 250 ng template DNA, and primer (50 pM for ERlC2, 50 pM for M13, or 24 pM for OPS-19) in a 50-^L final volume.

4. Program the thermal cycler and run the PCR as described in Table 4.

5. Prepare 2% (w/v) molecular grade agarose gels in 1X TBE buffer, load the samples and DNA marker, and run the electrophoresis for 1 h, 30 min at 150 V.

6. Dilute the stock solution of ethidium bromide to reach a concentration of 0.5 ^g/mL. Incubate the gel in the dark for 40 min. Wash the gel with distilled water and photograph the gel under 254-nm UV transillumination.

7. Interpret the banding patterns visually or with the help of a computerized analyzer (see Note 6).

Table 3

PFGE Electrophoretic Conditions for Some Salmonella Serotypes

Restriction endonuclease








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