31 Emerging Bacterial Foodborne Pathogens and Methods of Detection 729to trace the source of a L. monocytogenescontami-
nation in a food plant over a 6-month period. By tak-
ing samples over this time, culturing the isolates,
and performing RAPD analysis on individual iso-
lates, Lawrence et al. (Lawrence and Gilmour 1995)
showed that two strains were present throughout the
yearlong study. They showed that the strains were
persistent and might be responsible for cross-
contamination of the preparation areas within that
environment, and they were able to pinpoint the
source of the contamination of these strains. These
authors also demonstrated that other strains isolated
over time were transient in nature, and probably
came from various sources of contamination
(Lawrence and Gilmour 1995). This method is good
for microbial source tracking and determining criti-
cal control points for preventing pathogen contami-
nation within the food processing industry.
Ribotyping
Ribotyping is similar to RFLP in that it uses restric-
tion endonuclease digestion of DNA to set a pattern
that can be analyzed. This technique relies specifi-
cally on the ribosome-encoding genes that are rela-
tively conserved across the bacterial kingdom, and
allows lineages to be traced through the appearance
of mutations over time. To perform the procedure,
genomic DNA from individual strains is first digest-
ed with an endonuclease such as EcoRI, followed by
Southern hybridization with a probe targeted at spe-
cific conserved regions of rRNA coding sequence.
The probe allows only the DNA fragments encoding
rRNA to be detected on the blot (Ryser et al. 1996,
Weidmann et al. 1997). Different strains will give
rise to different banding patterns that are used to
determine lineages.
Weidmann et al. (1997) used ribotyping on L.
monocytogenesfrom a number of different isolates
to determine the different lineages related to the
pathogenicity of the organism. Using ribotyping, it
was possible to show that of three distinct lineages,
only one was responsible for all the human illnesses
(Weidmann et al. 1997).
Pulsed-Field Gel Electrophoresis
Pulsed-field gel electrophoresis allows for the sepa-
ration of large fragments of DNA, from 10–2000
kilobases (kb; Finney 2000). DNA fragments have
an overall negative charge proportional to their sizes,
due to the phosphate moiety on each nucleotide.
When an electric current is applied to a DNA sam-
ple, the DNA moves towards the positive electrode
(anode). Because the matrix of the gel acts as a
sieve, larger pieces of DNA are retarded in their
movement, while smaller fragments travel through
the spaces and migrate further on the gel. In a nor-
mal agarose gel, the largest size of DNA that can be
effectively separated is between 20 and 40 kb.
Above this size, limiting mobility occurs because
DNA fragments, once molded into a shape that can
squeeze through the sieve, migrate at around the
same rate.
Pulsed-field gel electrophoresis takes advantage
of the time it takes for the large DNA fragments to
squeeze into their elongated shapes for movement as
a basis for further separation. Larger DNA frag-
ments take longer to be forced into these shapes.
Therefore, if the direction of the electric field is
changed, then the smaller pieces of DNA will alter
their shape faster and start to migrate at the limiting
mobility rate. By optimizing the times and angles of
this alternating electrophoretic field, the larger
pieces of DNA can be resolved on the gel (Finney
2000, Moore and Datta 1994).
Pulsed-field gel electrophoresis can be used for
genetic fingerprinting of any pathogen isolate, al-
lowing for the determination of the relatedness
between cases of illness such as listeriosis (Don-
nelly 2001, Moore and Datta 1994). The U.S. CDC
initiated a collaborative effort to create an electronic
database known as PulseNet that encourages re-
searchers to enter their data to help track outbreaks
of E. coliO157:H7, nontyphoidal Salmonellasero-
types, L. monocytogenes,and Shigella(Swaminthan
et al. 2001). As of 2002, 46 state labs, two public
labs, the FDA, the USDA, and several Canadian labs
have entered data into PulseNet. This allowed for
better tracking and earlier detection of possible
common-source outbreaks (Kathariou 2002, Swam-
inthan et al. 2001). Although this method is finding
increased use and provides a great deal of informa-
tion, it is time consuming, requiring about 3 days to
obtain results (Finney 2000).FISH—Fluorescence In Situ HybridizationFluorescence in situ hybridization (FISH) is often
used to study, in a cultivation-independent way, the