2. For detection of tet(C), tet(D), tet(G), tet(Y), and tet(30), perform a two-step PCR amplification consisting of initial denaturation at 94°C for 5 min followed by 25 cycles at 94° for 5 s and 10 s of annealing/extension at 68°C, with a final extension at 68°C for 7 min. Include positive (Table 2) and negative (e.g., a noncomplementary template) controls for each pair of primers used.

3. For detection of tet(A), tet(B), tet(E), tet(H), tet(J), and tet(Z), perform a two-step PCR amplification consisting of initial denaturation at 94°C for 5 min followed by 25 cycles at 94° for 5 s and 30 s of annealing/extension at 61 °C, with a final extension at 61 °C for 7 min. Include positive (Table 2) and negative (e.g., a noncomplementary template) controls for each pair of primers used.

4. A second, nested PCR can be performed using 1 ^L of product from the first PCR as a template and amplifying for 25 cycles as just described if amplification fails owing to the presence of unidentified PCR-inhibiting substances.

5. Analyze the PCR products by electrophoresis of 5-^L aliquots on a 2.5% (w/v) agarose gel (NuSieve®, FMC Bioproducts) containing the fluorescent dye GelStar® (FMC Bioproducts). If running the gel without the dye, stain it in the ethidium bromide or GelStar solution after the run. The expected amplicon sizes are shown in Tables 1 and 2.

1. Perform PCR reaction as described earlier for the RPP and TEPGNB genes, but using a primer pair with a GC-clamp on one primer (see Note 3).

2. Analyze a 5-^L aliquot by agarose gel electrophoresis to confirm that sufficient quantities of the desired amplicon are synthesized.

3. Assemble the vertical gel casting module of the Bio-Rad D-Code System (Hercules, CA) including a GelBond PAG gel supporting film.

4. Form a 15-60% urea/formamide gradient (100% denaturant is equivalent to 7 M urea and 40% deionized formamide) in a 8% polyacrylamide gel (0.5X TAE buffer) using a BioRad Gradient Former.

5. Insert the comb and allow the gel to polymerize for 2 h.

6. Remove the comb and wash the pockets with the electrophoresis buffer.

7. Apply samples (1-10 ^L), mount the gel module in the preheated electrophoresis tank of the Bio-Rad D-Code System, and perform electophoresis at 60°C and 150 V for 2 h followed by 200 V for 1 h.

8. After the run, remove the supporting film with the gel on it, rinse in ddH2O, and fix in a solution of 10% ethanol and 0.5% acetic acid for 4 h to overnight with gentle shaking.

9. Rinse gel briefly in ddH2O.

10. Shake gently in a solution of freshly prepared silver stain solution for 20 min.

11. Rinse gel briefly in ddH2O.

12. Incubate the gel in developing solution with gentle shaking until the desired intensity of bands is obtained.

13. Rinse gel in ddH2O.

14. The gel can now be photographed or scanned. We usually capture and digitize the gel images using the Bio-Rad system, which includes a GS-710 Calibrated Imaging Densitometer connected to a G3 Macintosh computer with the Diversity Database™ software. Images can be saved in formats compatible with a number of other image software such as Adobe® Photoshop®.

3.5. Quantitative Real-Time PCR of TEPGNB Genes

1. Amplify the control templates using the corresponding primer pairs (see Subheading 3.3., Tables 2 and 3, and also Notes 4 and 5).

2. Run a preparative agarose gel (TAE buffer, 0.8% agarose) electrophoresis with the whole PCR mix applied.

3. Stain the gel with ethidium bromide.

4. Excise the band from the gel corresponding to the expected size amplicon while viewing under long-wave UV light.

5. Extract DNA with the QIAquick Gel Extraction Kit.

6. Determine the molar concentration of the purified DNA spectrophotometrically. Serial dilutions of this DNA will be amplified in parallel with the experimental samples using a real-time PCR thermocycler and will be used for generating standard curves to quantify the corresponding target genes in experimental samples.

7. For each sample: to QuantiTect SYBR Green master mix, add primers (at final concentration of 5 mM) and template, and then add RNase-free water from the kit to a final volume of 100 ^L. Distribute 25-^L aliquots to four tubes in a 96-well PCR plate.

8. Prepare other samples and control mixes as described in step 7. Seal the plate and perform PCR with initial denaturation/enzyme activation at 94°C for 10 min, followed by 45 cycles of two-step PCR consisting of 15-s denaturation at 94°C and 60-s annealing/extension at 61°C.

9. After the run, perform control analyses such as melting curve analysis and, if necessary, analyze the samples by agarose gel electrophoresis to verify the amplicon sizes.

10. Quantify the target gene concentration in experimental samples based on standard curves generated from the control tet templates.

4. Notes

1. Some DNA preparations obtained with the Mo Bio kits may still contain the substances inhibitory to PCR. If the concentration of your genomic DNA preparation is sufficiently high, then the sample may be diluted to decrease the concentration of these substances. Additional DNA purification may include phenol-chloroform-isoamyl alcohol (25:24:1) extraction and gel filtration. As a control for the suitability of DNA for PCR amplification, a PCR reaction with the universal bacterial primers 27f (5'AGAGTTTGATCM TGGCTCAG) and 1525r (AAGGAGGTGWTCCARCC) (44) can be performed.

2. In some cases, PCR amplification directly from samples (e.g., microbial isolates, tissue, and other biomasses) may be used, omitting the DNA extraction step. In our hands, successful amplification was done with the fresh colony biomass of laboratory Escherichia coli strains, Streptococcus spp., Enterococcus spp., Salmonella spp., and lactic acid bacteria. For this, resuspend one-half of a 1-2-mm individual colony in 20 ^L of sterile water and use 1-2 ^L of this suspension as a template for PCR amplification. The initial dena-turation time at 94°C should to be extended to 10 min to allow better denaturation of cellular proteins and more complete release of genomic DNA into a PCR mix. This labor-and time-saving shortcut may be especially important when a large number of samples need to be examined.

3. The DNA markers for PCR-DGGE of tetracycline resistance genes can be generated from control strains (Table 3).

4. Real-time PCR is extremely sensitive, allowing detection of several target molecules in a reaction, and care should be taken to reduce the possibility of contamination during all steps, beginning with purity of reagents, preparation of genomic DNA, and mixing of the PCR components. First, all solutions, water, and equipment for genomic DNA isolation must be free of tetracycline resistance gene contamination. Many molecular biology labs perform routine cloning with plasmids and strains that possess tetracycline resistance markers, the most widely used being tet(C), tet(B), and tet(A). These genes may circulate in the lab environment and air and may contribute to false-positive signals in real-time PCR. All surfaces, pipets, tube stands, and so on have to be cleaned with 3% H2O2 and ethanol and then UV-irradiated. The use of aerosol-protected filter tips is highly recommended. Second, genomic DNA isolation from samples and control strains should preferably be carried out separately to avoid crosscontamination. Third, mixing the components for real-time PCR should, if possible, be performed in a designated UV-treated box.

5. Albeit suitable for conventional PCR and PCR-DGGE, the TetC primer set (Table 2) produced an additional low-molecular-weight amplicon in real-time PCR. Therefore, this pair cannot be recommended for quantitative real-time PCR.


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