722 Part VII: Food Safety
pathogen contamination, DNA purified from the
food sample is serially diluted and added to a con-
stant amount of competitor DNA. PCR is per-
formed, and the intensity of the pathogen gene’s sig-
nal is compared to that of the competitor DNA on an
agarose gel. The number of cells in the original sam-
ple can be estimated by the intensity of the full-
length PCR product (from the pathogen) as com-
pared with the intensity of the smaller, competitive
PCR product (Schleiss et al. 2003). The advantage
to this method over a number of other PCR methods
is that no expensive fluorophores or radioactive la-
bels are required to visualize the results (Choi and
Hong 2003). Choi and Hong (2003) used a variation
of competitive PCR in which the competitor frag-
ment had a restriction endonuclease site removed so
that after PCR and digestion with the endonuclease,
the competitor was visualized as a slightly larger
molecule. The authors also varied the amount of
competitor DNA added and kept the sample inocula-
tion constant in order to show that the could quanti-
tatively determine the number of L. monocytogenes
in artificially inoculated milk. According to Choi
and Hong (2003), the method took around 5 hours to
complete without enrichment and was able to detect
103 cfu/0.5 mL milk using the hlyAgene. The detec-
tion limit could be reduced to 1 cfu if culture enrich-
ment for 15 hours was conducted first.
PCR-based methods have several limitations. First,
the food matrix, which includes complex polysac-
charides in feces and fat and proteins in food sam-
ples, often interfere with PCR by inhibiting DNA
polymerase directly or by binding Mg^2 (inhibiting
PCR enzymes) (Fratamico and Strobaugh 1998).
The matrix-based interference necessitates the use
of DNA purification steps that add to both the cost
and the completion time of the method. Second, cul-
ture enrichment may be required to concentrate the
pathogen so that gene of interest can be detected.
Third, analysis on an agarose gel is labor intensive
when done on the large scale required by the food
processing industry. Finally, PCR only detects the
presence of DNA. This does not indicate whether
the pathogens are dead or alive. The food industry
must know whether the food represents a health haz-
ard, not whether the pathogen was present at one
point but was killed by the food processing method.
Several groups have developed alternatives to the
use of classical PCR for pathogen detection to re-
duce some of the limitations of PCR. As mentioned
before, PCR protocols are generally time consum-
ing, sometimes requiring bacterial enrichment from
food samples before DNA can be amplified. Some
researchers have tried to address this problem by
performing PCR and then using oligonucleotide
probes complementary to the gene of interest to cap-
ture the amplified DNA. This separates and concen-
trates the amplified DNA to allow detection of the
amplified signal. While Ingianni et al. (2001) were
able to detect L. monocytogenesin food samples
using complementary probes, they admitted that their
results were much more reliable after overnight cul-
ture enrichment in selective media. This increased
their detection time from one working day to two,
with a detection limit of 2–10 cells/g sample.
A second rate-limiting step for PCR methods is in
the need to analyze and detect the amplified DNA
product. When dealing with hundreds or thousands
of samples, the time for running agarose gels with
the DNA samples or performing DNA hybridization
assays becomes significant (Koo and Jaykus 2003).
Some groups have improved PCR sensitivity by
using fluorescent resonance energy transfer(FRET)-
based PCR (Koo and Jaykus 2003). In this method,
DNA is analyzed directly after PCR by measuring
the fluorescence signal (See Fig. 31.2). This system
works by having two DNA probes for the gene of in-
terest, one with a fluorescein label and the other with
a quencher label. During the annealing and primer
extension steps of the PCR, the fluorescein-labeled
oligonucleotide hybridizes to the gene of interest.
The hybridized probe is digested by the exonuclease
activity of the DNA polymerase as the polymerase
amplifies the gene. This digestion releases the fluo-
rophore from the probe. The probe with the quencher
label is short and will not anneal to the fluorescein-
labeled probe until after the PCR process is com-
pleted and the mixture is cooled to room tempera-
ture. At this point, any unused fluorescein-labeled
probe is quenched due to its hybridization with the
quencher probe, leaving only the free fluorophore
(Koo and Jaykus 2003; see Fig. 31.2 for more de-
tails). The resulting fluorescence is proportional to
the number of pathogens in the original sample and
obviates the need to run agarose gels for PCR prod-
uct detection. A single probe containing both the flu-
orophore and quencher can also be used (Cox et al.
1998), but the double label significantly increases the
cost of probe synthesis. This method, targeting hlyA,
provides a detection limit for L. monocytogenesof