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Fig. 1. Evaluation of quantitative PCR (qPCR) methods to quantify
Yersinia pestis
in host and vector tissues.
(A)
Comparison of qPCR and colony-forming unit (CFU)
methods to track the decline in numbers of an attenuated
Y. pestis
strain after intra-
dermal (ID) injection. Skin biopsies from individual mice were taken 0-7 days after
injection with 10
5
Y. pestis
KIM6
+
. Biopsies were triturated with a tissue homoge-
nizer and divided equally for qPCR and plate count assays.
(B)
Comparison of qPCR
and CFU methods to quantitate
Y. pestis
in infected fleas. Flea triturates (
see
step 4,
Subheading 3.1.2.
) were divided equally for qPCR and plate count assays.
(C)
Standard curve prepared for qPCR of
Y. pestis
in rat lymph nodes as described in
Subheading 3.1.4
.
3. TaqMan® primers and probe specific for the plasmid-borne plasminogen
activator (
pla
) gene of
Y. pestis
:
Pla Forward Primer: 5´-CAAATATATCCCCTGACAGCTTTACA-3´
Pla Reverse Primer: 5´-AAGCATTTCATGAGACTTTCCACTC-3´
Pla Probe: 5´-6FAM-TGCAGCCCTCCACCGGGATGC-TAMRA-3´
4. TaqMan® Universal PCR Master Mix (Part No. 4304437, Applied Biosystems).
5. MicroAmp® Optical 96-well reaction plates and optical adhesive covers (Part
No. N801-0560 and 4311971, Applied Biosystems).
6. RNA protect (Qiagen, Valencia, CA).
7. DNA purification kit [we used the Puregene Kit D-50KA (Gentra, Minneapolis,
MN) for flea and skin biopsy samples and the Qiaprep Spin Miniprep Kit
(Qiagen) for the lymph node samples].
8. Qiashredder spin-column homogenizers (Qiagen, used for skin samples); a slurry
of 0.1-mm glass beads (BioSpec Products, Bartlesville, OK) in sterile H
2
O (used
for flea samples); cell strainer, 70-μm nylon mesh (BD Falcon, Bedford, MA;
used for lymph node samples).
9. Plastic pestles that conform to the interior of a 1.5-mL microfuge tube.
10. Brain-heart infusion (BHI) broth (Difco, Sparks, MD) and sterile PBS.
11. 3% H
2
O
2
and 70% ethanol.
12. Petroff-Hausser bacterial counting chamber.
13. Spectrophotometer.
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