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

292 Wastling and Mattsson
2. Remove the tube from the centrifuge and using a plastic pipet, carefully remove
the interface containing the white cells. Mix this with 2 mL PBS.
3. Layer the diluted white cells over Lymphoprep (10 mL) and spin at 1000g for
15 min to clean the cells further. Again, carefully remove the white cell interface
using a plastic disposable pipet and decant it into a clean tube.
4. At this stage, some RBCs may remain, and these are best removed by lysis with
ammonium chloride. To do this, treat the sample with an equal volume of 10 mM
Tris-NH 4Cl for 2 min at room temperature and then wash the resulting cell suspension
3× by resuspension in PBS and centrifugation at 1000g for 10 min. The
pellet may now be stored frozen at –20°C for later processing if desired.
5. Resuspend the resulting pellet in 50 µL of 10 mM Tris, pH 8.3, 40 mM KCl,
2.5 mM MgCl 2 containing proteinase K 100 µg/mL and Tween 20 (0.5%).
6. Incubate the mixture at 55°C for 1 h and then inactivate the proteinase K by
boiling for 5 min.
7. Store the samples at –20°C for later PCR analysis (see Note 2).
3.1.2. Lymph
1. Dilute the lymph sample 1 in 10 with PBS to prevent clotting and centrifuge at
1000g for 10 min. Resuspend cells in 1 mL PBS.
2. Lyse any contaminating RBCs by the addition of 1 mL of 10 mM Tris-NH4Cl and
wash the remaining cells 3× by resuspension in PBS and centrifugation at 1000g
for 10 min. The pellet may now be stored frozen at -20°C for later processing.
3. Resuspend the resulting pellet in 50 µL of 10 mM Tris, pH 8.3, 40 mM KCl,
2.5 mM MgCl 2 containing proteinase K 100 µg/mL and Tween 20 (0.5%).
4. Incubate the mixture at 55ºC for 1 h and then inactivate the proteinase K by
boiling for 5 min.
5. Store the samples at -20°C for later PCR analysis (see Note 3).
3.1.3. Tissue Samples
PCR can be performed either on fresh or frozen tissue. However, if tissue is
to be frozen before analysis care should be taken to freeze samples at –20°C as
soon as possible after collection as autolysis of the sample and degradation of
DNA may occur quite rapidly.
1. For processing for PCR, finely chop the samples and wash with ice-cold PBS.
2. Digest the tissues overnight at 37°C in 100 µL of 50 mM Tris, pH 8.5, 1 mM
EDTA, Tween 20 (0.5%) containing proteinase K 200 µg/mL.
3. After digestion, inactivate the proteinase K by boiling for 5 min.
4. Store the samples at –20°C for later PCR analysis.
3.2. DNA Amplification
Detection of the parasite is by amplification of the T. gondii repetitive B1 locus.