Biology Reference
In-Depth Information
3. Methods
3.1. Preparation of Standard Curve
The standard curve is determined from a set of samples containing known
numbers of
Y. pestis
in uninfected tissue of the same type and amount as the
infected (unknown) samples. Both standard and unknown samples are then
processed identically for qPCR. Careful matching of the tissue background
of both standard and unknown samples cancels out the effects of any PCR
inhibitors inherent in a particular tissue type. Because the
pla
gene target
occurs on a plasmid of unknown but reportedly high copy number per cell,
the standards are based on numbers of bacteria rather than numbers of gene
target copies. This maintains the increased sensitivity provided by a multi-copy
target while permitting estimation of the absolute number of bacterial cells per
unknown sample.
3.1.1. Preparation of Stock Bacterial Standards
1. Grow
Y. pestis
KIM6
+
(or other attenuated,
pla
+
strain) in liquid BHI medium at
28 °C to stationary phase (overnight).
2. Determine the number of
Y. pestis
per mL by direct count, using a Petroff-Hausser
bacterial counting chamber and a phase-contrast microscope (400× magnification).
3. Centrifuge a known volume of culture to pellet the bacterial cells, remove the
culture supernatant, and resuspend the bacteria in sterile PBS to a concentration
of 1.0 × 10
9
per mL.
4. Aliquot this stock suspension and store the aliquots in a -80 °C freezer.
3.1.2. Preparation of Standard Curve Samples to Quantitate Y. pestis
in Fleas
1. Surface-sterilize uninfected fleas (one for each standard and one for the negative
control) by sequentially washing them for 1 min in 3% H
2
O
2
and 70% ethanol.
After the ethanol wash, allow the fleas to air-dry and rinse them in sterile distilled
H
2
O.
2. Thaw a stock bacterial standard aliquot, vortex, and make 1:10 serial dilutions in
sterile BHI broth. Six standards containing 1 × 10
2
to1×10
7
bacteria in 100 μL
BHI in a sterile 1.5-mL microfuge tube are needed, plus a negative control tube
containing 100 μL sterile BHI.
3. Add 20 μL of sterile glass bead slurry and an uninfected, surface-sterilized flea
to each of the six standards and the negative control.
4. Triturate each flea thoroughly by grinding it with a pestle into the glass beads
against the bottom of the microfuge tube. Vortex well, then allow glass beads and
flea debris to settle out before transferring the supernatant to a clean microfuge
tube. Proceed to DNA extraction (
see
Subheading 3.3.
).
Search WWH ::
Custom Search