31 Emerging Bacterial Foodborne Pathogens and Methods of Detection 727sample is suspected of harboring a pathogen. These
methods are not used as routine screening tools.
Each allows further characterization of the pathogen
in order to know which of the many possible sub-
types within a serotype is causing the disease or is
the contaminating factor. The information is impor-
tant from an epidemiological point of view and is
needed to identify the precise strains responsible for
causing disease, or that contaminate a certain type of
food. The main drawback with these methods is that
individual cultures have to be isolated and grown
before being analyzed independently. The methods
are compared in Table 31.4.
RFLP—Restriction Fragment Length
Polymorphism
Restriction fragment length polymorphism (RFLP)
analysis uses the restriction endonuclease patterns in
DNA to determine differences in genetic profiles.
Even among closely related individuals, the muta-
tion of a restriction site occurs often enough to allow
a pedigree to be formed (Smith and Nelson 1999).
Digesting genomic DNA using restriction endonu-
cleases creates fragments of different lengths. The
DNA is then run on a capillary electrophoresis and
the pattern of bands analyzed. Different groups of
organisms, or serotypes within the same species, can
be elucidated from the differences in the electro-
phoresis patterns of DNA fragments. However, di-
gesting genomic DNA often results in too many
bands, leading to difficulty in interpretation of re-
sults. To minimize interference, RFLP can be done
on individual genes. Weidmann et al. (1997) used
PCR-RFLP on the hlyA, actA,and inlAgenes of L.
monocytogenesto determine how strains with differ-
ent pathogenic potentials were related. They found
that L. monocytogenescould be divided into three
lineages, and that all epidemic outbreaks could be
traced back to lineage I.
AFLP—Amplified Fragment Length
Polymorphism
Amplified fragment length polymorphism (AFLP)
analysis is a DNA fingerprinting technique based on
the selective amplification of genomic restriction
fragments to generate a restriction pattern formed
from a large number of fragment bands on gels
(Aarts et al. 1999). It essentially combines the relia-
bility of RFLP and the power of PCR. Genomic
DNA is digested with two restriction endonucleases,
one of which cuts infrequently, and the other on a
frequent basis (Blears et al. 1998). Selective pres-
sure for amplification is then applied in two different
ways. The first is that adapter oligonucleotides are
ligated to the digested DNA. The adapters add a few
selective nucleotides after the restriction site. Only
those fragments with the complementary sequence
between the restriction site and the adaptor will be
bound and amplified by PCR. This step results in
about 1/16 of the bands being amplified during the
PCR (Aarts et al. 1999). After the PCR process, the
amplified fragments are separated from the mixture
by the presence of a biotin label on the adapter on
the infrequently cut end of the fragment. Those frag-
ments are separated from the bulk solution by the
use of streptavidin-coated magnetic beads (Blears et
al. 1998). Selective amplification results in 50–100
different fragments being amplified (Blears et al.
1998). Analysis is performed and the amplified frag-
ments detected by the presence of a fluorophore on
one of the primers. Detection of the fingerprinting
pattern can be done either on gels or by automated
laser fluorescence analysis. The latter allows com-
parison of data from other laboratories (Aarts et al.
1999). Genetic polymorphisms are then identified
by the presence or absence of different fragments
(Blears et al. 1998).
Aarts et al. (1999) used AFLP to differentiate dif-
ferent strains of L. monocytogeneson a more dis-
criminating basis than serotyping. In particular, the
results match those obtained by other methods such
as pulsed-field gel electrophoresis, multilocus PCR-
restriction enzyme digestion of the iapgene locus,
and ribotyping (Aarts et al. 1999).RAPD—Randomly Amplified Polymorphic
DNARandomly amplified polymorphic DNA (RAPD)
analysis makes use of a short arbitrary primer (e.g.,
10 bp) that anneals randomly along genomic DNA
to amplify a number of fragments within the gen-
ome. As long as the same primer is used for all the
test samples, the comparison of the number and
sizes of fragments generated allows for discrimina-
tion between strains of a pathogen. It does require
having a pure culture so that there are no contami-
nating bands from other organisms or from the DNA
in the food (Lawrence and Gilmour 1995). With
regard to L. monocytogenes, the technique was used