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

102 Ewalt and Bricker
Primer Cocktail from step 2, 500 µL GC Rich Enhancer (optional) (see Note 7).
(If an enhancer is not used, then 500 µl PCR-grade water should be substituted),
and 20 µL FastStart Taq DNA Polymerase.
4. Mix the solution thoroughly, but gently, by pipeting up and down (see Note 13).
5. Aliquot the Master Mixture in 25-µL quantities into 0.2-µL thin-walled PCR
tubes (or alternatively, a PCR-certified 96-well plate) (see Note 14). Store the
assay tubes at –20° 2°C.
6. Prior to use, thaw enough Master Mixture tubes for unknowns and controls and
mix thoroughly but gently by finger tapping.
3.3.2. Amplification of Products by PCR
1. Add between 1.0 and 2.5 µL of unknown sample or control to each assay tube
(see Note 15). Be sure to mix each sample thoroughly, just before removing the
aliquot, since Brucella tends to settle out quickly.
2. Amplify the PCR products by using the following parameters: 1 cycle of 95°C
for 5.0 min (see Note 16), then 40 cycles of 95°C for 15 s, 52°C for 30 s, 72°C for
1 min 30 s, and then 4°C indefinitely. The choice of ramp-time does not appear to
be critical.
3. After amplification, the unopened samples can be stored at 4°C until ready for
detection.
3.4. Detection of Amplified Products
1. Prepare a 5-mm thick 2.0% agarose gel (in 0.5X TBE) with an appropriate number
of wells (see Note 17).
2. Combine 1 µL of 6X loading dye with 8 µL amplified sample and mix well
before loading into the gel well (see Note 18).
3. Run the gel in 0.5X TBE until the bromophenol blue marker is at least 5 cm from
the well to achieve good separation of the bands. For the equipment described
here, 80–85 V for 2.5 h maximizes resolution without significant diffusion of the
amplified DNA bands. Adjustments in voltage and time may be needed for other
brands of equipment.
4. Stain the gel for 45 min in ethidium bromide solution (250 µg/500-mL of 0.5X TBE).
Alternatively, the gel can be stained before electrophoresis or during electrophoresis
by adding ethidium bromide to the running buffer (see Note 19). CAUTION:
ethidium bromide is a mutagen and potential carcinogen (see Note 20).
3.5. Interpretation of Data
Identification is based on the number and the sizes of the products amplified
by PCR (see Fig. 1). All samples except the negative controls should amplify at
least 1 DNA product, the 800-bp 16S sequence. If this band is not present, then
the sample may contain PCR inhibitors, the DNA was degraded, or the sample
was not dispensed into the Master Mixture. It may be necessary to dilute the
original sample to decrease the level of inhibitors in the reaction, repeat the assay
with a fresh sample, or simply repeat the assay with the original sample.