BLBS102-c44 BLBS102-Simpson March 21, 2012 14:34 Trim: 276mm X 219mm Printer Name: Yet to Come
44 Emerging Bacterial Food-Borne Pathogens and Methods of Detection 845Polymerase Chain Reaction (PCR)PCR has revolutionized molecular studies and our approaches
in detection and characterization of food-borne pathogens. The
basic principle of the technique is that a thermostable DNA
polymerase is used to amplify a specific region of DNA to de-
tectable levels from template DNA found in the target pathogen.
In conventional PCR methodology, the amplicons generated are
then separated out by gel electrophoresis, the DNA stained, and
the size of the DNA bands determined by comparison to stan-
dard DNA and a control strain. The use of such amplification
techniques has expanded considerably over recent years and is
used not only in the detection of a target pathogen but also for
identification of pathogens in foods and other substrates, detec-
tion of genes associated with pathogenesis, virulence or toxins,
and detection of viruses or fungi (Auvray et al. 2007, Kim et al.
2007b, Baert et al. 2008, Niessen 2008).
Newer PCR techniques are emerging, making this technique
ever more useful for rapid assessment of a food. For example,
PCR reactions are commonly configured to amplify multiple
gene targets simultaneously; therefore, the application of PCR-
based approaches has the capability to detect multiple pathogens
concurrently. To work well, multiplex PCR protocols should use
primer sets which ensure that amplification products are at least
30-50 bp apart, thus allowing adequate resolution if the am-
plification is followed by gel electrophoresis. Multiplex PCR
reactions have been used in a number of ways including the
simultaneous detection of multiple target pathogens in a mixed
sample or matrix, for example, the detection of pathogens such
asSalmonella,Shigella,Listeria, E. coli O157:H7, and so on
in artificially inoculated foods (Alarcon et al. 2004, Kim et al.
2005, Kim et al. 2007b, Yuan et al. 2009, Kawasaki et al. 2010)
and detection of multiple virulence or resistance genes inE. coli
andSalmonella(Skyberg et al. 2003, Skyberg et al. 2006, Li
et al. 2006, Johnson et al. 2007, Nde and Logue 2008). Mar-
tinez et al. (2006) used multiplex PCR for the simultaneous
detection of three genes associated with the production of cyto-
lethal distending toxin (CDT) inC. jejuniand found the preva-
lence of the genes to be 98% in human and animal isolates
recovered from a wide European geographic origin. In a sim-
ilar study, Asakura et al. (2008) developed a multiplex forcdt
genes inC. jejuniandC. coli, with a minimum detection limit of
10–100 CFU.
When PCR is used in the detection of target genes from food
matrices or mixed culture, the quality of the DNA that serves
as template for amplification is of concern. Cultures and food
matrices can contain substances which inhibit PCR (Fakhr et al.
2006); these include components such as carbohydrates, fats,
polysaccharides and proteins; commercial PCR clean-up kits are
available for reducing these effects. The efficiency of, or utility
of PCR may be enhanced using different enzymes and labeling
agents; specialtyTaqpolymerases can increase the efficiency of
the PCR reaction (Fratamico and Bales 2005), and fluorescent-
labeled primers has been shown to lower detection limits. Chen
et al. (1998) used fluorescent-labeled primers to detect STEC in
foods. Primers were designed to detect the presence of the shiga
toxin genesstx1,stx2, andstxe. Detection limits of the methodwere 1-5 CFU per PCR mixture (pure culture), and 3 CFU per
25g of food. Fanning et al. (1995) used a similar approach to
develop a color amplified PCR system to detect the heat stable
toxin gene (ST) in enterotoxigenicE. coli(ETEC). The method
was found to have a lower detection limit of 10 fg of purified
DNA and was capable of detecting 270 CFU of an ETEC strain
possessing the ST gene.
Niessen (2008) described the use of PCR for the diagnosis
and quantification of mycotoxin-producing fungi in foods and
other commodities, while Zhang et al. (2008) reported a modi-
fication of PCR using an immuno-PCR assay for the detection
of shiga toxin 2 (stx2) in culture which reduced the sensitivity
to a level of 10pg/ml, when compared with commercial enzyme
immuno assays which had a limit of detection of 1 ng/mL. Ge
et al. (2002) used a combination of PCR with ELISA to de-
tect STEC in food and found detection limits of the assay were
0.1–10 CFU, depending on the strain type. Another modifica-
tion of PCR combined restriction fragment length polymorphism
(RFLP) (Atanassova et al. 2001) for the detection ofS. aureus
and staphylococcal enterotoxins in pork and pork products. This
method was found to be more sensitive than standard culture
techniques with a detection rate of 28.6–34.8% compared to
11.1% by standard culture. Other modifications of PCR include
restriction site specific PCR (RSS-PCR) (Kimura et al. 2000)
and the ramification assay for detection ofE. coliO157:H7 and
STEC (Li et al. 2005).Nested PCRNested PCR is a refined method for enhancing the specifics of
PCR for pathogen detection. It uses a primary set of primers
to amplify a specific region of DNA, then a secondary set of
primers is used to amplify a product that lies (i.e., nested) within
the initial product. Nested PCR is designed to increase the accu-
racy of the PCR reaction; that is, the dual amplification strategy
ensures that if the wrong target is amplified in the primary reac-
tion, the odds of a second successful amplification is low, as the
second pair of primers are smaller and designed to amplify only
within a region of the first PCR product. Therefore, the odds
of amplifying such a product in an error sequence become low
and the accuracy of the results are likely increased over standard
methods. Nested PCR has been used in the detection ofShigella
in food (Lindqvist 1999, Warren et al. 2006) and for the detec-
tion of toxin-associated genes in some pathogens. Miwa et al.
(1996) described the use of nested PCR for the detection of en-
terotoxigenicClostridium perfringensin animal feces and meat
using a pair of nested primers homologous to theC. perfringens
enterotoxin gene. The sensitivity of the test was about 10^3 fold
greater than standard PCR.Real-time PCRReal-time PCR is a modification of conventional PCR that elimi-
nates the need for gel electrophoresis, thus providing the user re-
sults more quickly. A recent review by Mackay (2004) provides