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

Campylobacter PCR 119
10. Once cell suspensions are prepared, the tubes should go immediately into the
boiling water bath. If this is not practical, as when preparing large numbers of
samples, keep the tubes on ice until ready to be boiled.
11. The boiled cell suspension/DNAzol mixture can be held overnight at 4°C if necessary.
All steps in the DNA extraction procedure are carried out at room temperature
unless otherwise noted.
12. All components, except the water, should be stored frozen at –20°C. The dNTP
mixture and the working primer stocks should be aliquoted to avoid repeated
(5X) freeze-thaw steps. Once the Taq DNA polymerase has been added, avoid
letting the master mixture sit for any length of time, as this may promote the
generation of nonspecific PCR products. Ideally, the PCRs should be assembled
in isolation, such as in a biocontainment hood, to minimize the possibility of
contaminating the reactions. A set of pipetors dedicated to PCR work should be
used in conjunction with aerosol-resistant pipet tips as a further means of reducing
contamination.
13. Primer sets are usually shipped in lyophilized form from the manufacturer. Prepare
a 400 µM stock solution of each in TE buffer, pH 7.5. This calculation is
easily made using the yield in nanomoles or micromoles for each primer provided
by the manufacturer. Using the stock solutions, prepare 1:20 dilutions in
water of each primer for the 20 µM working solutions. Water is used as the diluent
to minimize the amount of EDTA (which chelates the divalent magnesium ions)
added to the PCR.
14. DNA template sample vol can range from 0.5–10 µL, depending on the concentration
of DNA. The vol of water to add in the 1X PCR formula must be adjusted
accordingly; 5 µL is a convenient volume to accurately pipet.
Control tubes should always include a negative control in which water is substituted
for the sample DNA template. In this way, contamination of the PCR mixture
or nuclease-free water can be detected. A positive control using known
template material serves as a check on the PCR.
Acknowledgments
The authors thank the technical staff of the Antimicrobial Resistance
Research Unit, especially Sandra House, Leena Jain, Jennifer L. Murphy, and
Jodie Plumblee.
References
1. Friedman, C.R., Neimann, J., Wegner, H. C. and Tauxe, R.V. (2000) Epidemiology
of Campylobacter jejuni Infections in the United States and other Industrialized
Nations in Campylobacter, 2nd ed., (Nachamkin, I. and Blaser, M. J., eds.),
ASM Press, Washington, DC, USA, pp. 121–138.
2. Jacobs-Reitsma, W. (2000) Campylobacter in the food supply in Campylobacter,
2nd ed., (Nachamkin, I. and Blaser, M. J., eds.), ASM Press, Washington, DC,
USA, pp. 467–481.