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

110 Englen et al.
which campylobacters are intrinsically resistant, are also added to inhibit competing
bacterial and fungal flora found in the sample material (9). The selective
plating medium we prefer, Campy-Cefex (10), is described below. Campy-
Cefex plates are relatively translucent, and Campylobacter colonies are easier
to identify and quantify compared to media formulations containing charcoal,
such as the widely used modified charcoal cefoperazone desoxycholate agar
(mCCDA). However, mCCDA (available from Oxoid, Ogdensburg, NY,
USA), or the recently developed Campy-Line agar (11), may be directly substituted
for Campy-Cefex in the protocols outlined below.
Over the last decade, the polymerase chain reaction (PCR) has become a
basic tool for the identification of bacterial pathogens such as Campylobacter
(12,13). For those unfamiliar with the use of PCR for the amplification of specific
DNA sequences, a number of reviews are available (e.g., 14–16). Beyond
the basic requirement of suitable DNA sample preparations to serve as template
in the PCR, a specific DNA amplification (target) sequence must be
determined. Although usually a well-characterized region of the genome, this
is not always a requirement for the development of a useful assay (17). The
range of genes reported for the identification of Campylobacter spp. by PCR
includes 16S rRNA (18,19), 23S rRNA (20), the cadF virulence gene (21), and
the flagellin genes, flaA and flaB (22,23). The PCR we describe here is based
on the hippuricase gene (hipO) for the identification of C. jejuni (24) and a
siderophore transport gene (ceuE) sequence to identify C. coli (25). A separate
PCR is run for each primer pair. This PCR has also been adapted to allow the
direct identification of C. jejuni from the sample material without prior enrichment
steps (26). For applications involving large numbers of samples, multiplexing
(27) can be incorporated to reduce the total assay time and increase
sample throughput.
Finally, it is important to note that some strains of Campylobacter are resistant
to lysis by heating (28) and simply boiling cell suspensions for use in PCR
may result in a significant percentage of false negatives. The DNA isolation
procedure included here provides a simple method for consistently obtaining
usable template DNA from Campylobacter.
2. Materials
2.1. Isolation and Culture of Campylobacter
1. Exam gloves or gloves and fecal loops (Revival Animal Health, Orange City, IA, USA).
2. Sterile tongue depressors.
3. Sterile cotton-tipped swabs.
4. Sterile spreader sticks (Simport Plastics, Beloeil, Quebec, Canada).
5. Whirl Pak bags (Nasco, Fort Atkinson, WI, USA).
6. Gallon zip-lock bags (268 × 279 mm).