A PCR-based method to simultaneously detect and type
Mycobacterium tuberculosis complex bacteria
A. In vitro amplification of spacer DNA by PCR
Amplification of the spacers is accomplished by using the primers DRa and DRb, which enable to amplify the whole DR region. Only a very small amount of template DNA is required. Typically the PCR is performed on 10 ng purified chromosomal Mycobacterial DNA but, with minor adaptations, DNA extracts from clinical samples or lysed bacteria (from freezer or Löwenstein) can also serve as template. The PCR products are labeled with biotin, because primer DRa is biotinylated.
- Always include chromosomal DNA of M. tuberculosis strain H37Rv and M. bovis BCG P3 as positive controls. Use water as a negative control.
- Prepare the reaction mixture:
2 μl template DNA
3 μl primer DRa (0.2 μmol/μl)
3 μl primer DRb (0.2 μmol/μl)
20 μl 2×TaqPCR MasterMix
12 μl MQ water (to a final volume of 40 μl)
- Place the tubes in a PCR-apparatus for amplification, and perform the following temperature cycling:
B. Hybridization with PCR product and detection
Hybridization of the biotin-labeled PCR products to the immobillized spacer-oligos that represent spacers of known sequence. The presence of spacers is visualized on film as black squares after incubation with streptavidin-peroxidase and ECL-detection.
- All buffers should be prewarmed before use. Prepare the following buffers from concentrated stocks, using demineralized water for dilution (quantities for one membrane):
2×SSPE/0.1 % SDS, 42 °C,
2×SSPE/0.5 % SDS, 60 °C,
2×SSPE/0.5 % SDS, 42 °C.
2×SSPE, room temperature.
- Add 25 μl of the PCR products to 150 μl 2×SSPE/0.1 % SDS.
- Heat-denature the diluted PCR product for 10 min at 100 °C and cool on ice immediately.
- Wash the membrane for 5 min at 42 °C in 250 ml 2×SSPE/0.1 % SDS.
- Place the membrane and a support cushion into the miniblotter, in such a way that the slots are perpendicular to the line pattern of the applied oligonucleotides.
- Remove residual fluid from the slots of the miniblotter by aspiration.
- Fill the slots with the diluted PCR product (avoid air bubbles!) and hybridize for 60 min at 60 °C on a horizontal surface (no shaking!). Avoid contamination of neighbouring slots.
- Remove the samples from the miniblotter by aspiration and take the membrane from the miniblotter using forceps.
- Wash the membrane twice in 250 ml 2×SSPE/0.5 % SDS for 5 min at 60 °C.
- Place the membrane in a rolling bottle and allow it to cool down to prevent inactivation of the peroxidase in the next step.
- Add 5 μl streptavidin-peroxidase conjugate (500 U/ml) to 14 ml of 2×SSPE/0.5 % SDS, and incubate the membrane in this solution for 60 min at 4 °C in the rolling bottle.
- Wash the membrane twice in 250 ml of 2×SSPE/0.5 % SDS for 10 min at 42 °C.
- Rinse the membrane twice with 250 ml of 2×SSPE for 5 min at room temperature.
- For chemiluminiscent detection of hybridizing DNA, incubate the membrane for 1 min in 16 ml ECL detection liquid.
- Cover the membrane with a transparent plastic sheet or Saran-wrap and expose a light sensitive film to the membrane for 20 min.
- If the signal is too weak or too strong the membrane can be used again directly to expose another film for a shorter or longer period.
C. Regeneration of the membrane
The hybridized PCR product is dissociated from the membrane in order to regenerate the membrane for the next hybridization. A membrane can be regenerated for at least 10 times.
- Wash the membrane twice by incubation in 1 % SDS at 80 °C for 30 min.
- Wash the membrane in 20 mM EDTA pH 8, for 15 min at room temperature.
- Store the membrane at 4 °C until use (sealed in plastic or wrapped in Saran-wrap, to avoid dehydration of the membrane).