PCR Detection of Microbial Pathogens PCR Detection of Microbial ...

254 Kobisch and Frey
First round of amplification:
1. Use thin-walled thermal cycler tubes precooled at 4°C.
2. Add for each first-round reaction: 45.0 µL PCR premixture with corresponding
primers, 0.25 µL Taq DNA polymerase, and 5.0 µL template.
3. Preheat thermal cycler to 95°C.
4. Place the cooled tubes directly into preheated thermal cycler.
5. Run 35 cycles with: 30 s at 94°C, 30 s at annealing temperature (according to
Table 1), and 45 s at 72°C.
Second round of amplification:
6. Prepare the second round in a precooled thin-walled tube by mixing: 45.0 µL
PCR premixture with corresponding primers, 0.25 µL Taq DNA polymerase,
and 1 µL amplicon of the first round.
7. Preheat thermal cycler to 95°C.
8. Place the cooled tubes directly into the preheated thermal cycler.
9. Run 35 cycles with: 30 s at 94°C, 30 s at annealing temperature (according to
Table 1), and 30 s at 72°C.
10. Take the tubes to a different room for analysis.
11. Proceed to either direct visualization of the PCR products on agarose gel electrophoresis
(see Subheading 3.4.3.) or Southern blot hybridization with a radioactively
labeled probe (see Subheading 3.4.4.).
3.4.3. Direct Visualization of PCR Products on Agarose Gels
1. Analyze 10 µL of each PCR amplicon on a 0.7% agarose gel.
2. Stain the gel with ethidium bromide and photograph fluorescence by UV light.
3. Analyze the correct sizes of the bands (see Table 1) and verify all negative and
positive controls.
3.4.4. Analysis of PCR Products by Southern Blot Hybridization
In order to insure a very high sensitivity of the nested PCR method with
primer pairs Hp1/Hp3 and Hp4/Hp6, which is used preferentially for the
amplification of samples from tracheobronchiolar washings, the products of
the second PCR amplification are submitted to agarose gel electrophoresis,
followed by transfer on a nylon membrane and hybridization with the radioactively
labeled primer Hp43.
3.4.4.1. LABELING OF THE OLIGONUCLEOTIDE PRIMER
1. Prepare kinase reaction for labeling primer Hp43: 2 µL (20 pmol) Oligonucleotide
primer Hp43 (10 µM), 5 µL 10X Kinase buffer, 5 µL (total ATP minimal
1 µM) [γ- 32 P]ATP (1000 Ci/mmol, 10 µCi/µL), 2 µL bacteriophage T4-polynucleotide
kinase (10 U/µL), and 46 µL H 2O.
2. Incubate 30 min at 37°C.