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

236 Hotzel et al.
3. Methods
3.1. DNA Extraction from Different Sample Matrixes
3.1.1. Broth Culture
The simplest method to release DNA suitable for PCR from broth cultures
of mycoplasmas is 5-min of boiling. After removal of cellular debris by centrifugation
at 12,000g for 1 min, the supernatant can be used directly as template.
Failure to amplify a specific target could be due to the presence of PCR
inhibitors. In these instances, a commercial DNA extraction kit should be tried
(see Note 1).
3.1.2. Swabs from Nasal Mucus, Conjunctival,
Pleural, Synovial, or Bronchial Lavage Fluid
1. Pipet 500 µL of lysis buffer into a 2-mL Safe-Lock tube containing the cotton
swab.
2. Vortex mix thoroughly for 1 min.
3. Centrifuge at 12,000g for 30 s.
4. Put the swab into a 1-mL pipet tip whose lower half was cut off and place it all
into a fresh tube.
5. Centrifuge at 12,000g for 1 min to force the remaining liquid out of the cotton.
6. Add the liquid to that in the first tube from step 3.
If you have mucus or fluid samples (no swabs), start with step 7.
7. Centrifuge the liquid or mucus at 12,000g for 15 min.
8. Discard the supernatant and resuspend the pellet in 50 µL of lysis buffer.
9. Add 20 µL of proteinase K and incubate at 60°C for 2 h.
10. Inactivate the proteinase K by heating at 97°C for 15 min.
11. Centrifuge at 12,000g for 5 min to remove debris.
12. Use 5 µL of the supernatant for PCR.
3.1.3. Milk Samples for the Detection of M. bovis
The following protocol includes pre-PCR enrichment by antigen capture and
subsequent DNA extraction and is recommended for milk samples containing
M. bovis (see Note 2).
3.1.3.1. COATING OF MICROTITER PLATES
1. Pipet 100 µL of the solution containing MAb 4F6 in CBB into the cavities of a
96-well microtiter plate.
2. Incubate overnight at 4° C.
3. Rinse 3× with 200 µL of PBS per well and empty the wells.
4. The plates can now be sealed with parafilm and stored at –20° C until use.
3.1.3.2. SAMPLE PROCESSING
1. Introduce 200 µL of milk samples into the wells of a MAb-coated microtiter plate.