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846 Part 8: Food Safety and Food Allergensgood information into the detail of the technology. Whereas, its
underlying principle is based on standard PCR, a fluorescent tag
is added to the primers so that the amplicons can be detected on
a real-time basis by monitoring fluorescence. Thus, a detector
in the thermocycler detects amplified product as it is produced.
Real-time PCR protocols can be designed to detect multiple
products simultaneously using a similar approach to multiplex
PCR—typically, four or five fluorescent tags can be detected si-
multaneously as with conventional multiplex PCR. It also means
that products of the similar size can be amplified easily without
the challenges posed by separation on a standard electrophoresis
gel, as each amplicon will be amplified with a different fluores-
cent marker. The advantages of real-time PCR over conventional
PCR include: easy resolution of product, a faster run time as there
is no post-PCR gel analysis, and increased sensitivity. The latter
is greater than conventional PCR, as real-time PCR technology
relies on the detection of a signal and its quantification with the
release of light being in proportion to amplified product formed.
Fluorescent dyes such as HEX, FAM, ROX, and SYBR green
are some of the commonly available fluorescent dyes, which can
be used simultaneously in a multiplex real-time-PCR protocol
(Huang et al. 2007, Wang et al. 2007, Nde et al. 2008b). Multiple
commercial thermocyclers are available for use in performing
real-time PCR offering capabilities from single tubes to 96 and
384 well formats and have the capability to detect up to five dif-
ferent fluorescent targets simultaneously. Despite the practical
advantages of this method, the expense of the requisite thermo-
cyclers is high that may curtail the use of real-time PCR in some
instances. Potential applications of real-time PCR include detec-
tion of genes or strains in a range of media (Rodriguez-Lazaro
et al. 2004, Fakhr et al. 2006, Bohaychuk et al. 2007, Wang et al.
2007, O’Grady et al. 2008), and diarrheagenicE. coli(Vidal
et al. 2005). Horsmon et al. (2006) used real-time fluorogenic
PCR to detectentA, encoding the staphylococcal enterotoxin A
(SEA). The method detected SEA at levels as low as 1–13 gene
copies. Fykse et al. (2007) used molecular beacon real-time nu-
cleic acid sequence-based amplification forVibrio choleraeby
detecting the cholera toxin gene (ctxA) and the genestcpA,toxR,
hlyA, andgroELand was able to detect the organism at a level
of 50 CFU/mL, the method could also differentiate toxigenic
from nontoxigenicVibriostrains by amplification of the toxin
genestcpAandctxA.Grant et al. (2006) modified the real-time
PCR technique to simultaneously detect heat stable and labile
toxin genes of enterotoxigenicE. coliwith threshold cycles of
25.2–41.1.
A primary limitation of PCR has been that it could not dis-
tinguish between live and dead cells. However, newly designed
PCR protocols can target viable cells by detection of mRNA,
which is a marker of viability (Klein and Juneja 1997, McIng-
vale et al. 2002, Morin et al. 2004).
Overall, PCR has tremendous power as a molecular detection
and profiling tool especially as a means to detect a pathogen as
well as determine a pathogen’s traits or to define its pathotype. It
can also be useful for clustering gene traits and can provide sig-
nificant information when bundled with analysis software to sort
organisms into cluster groups or by trait possession (Rodriguez-
Siek et al. 2005a, 2005b).NEXT-GENERATION TECHNOLOGIES
The application of sensitive and rapid detection technologies has
become of paramount interest in advancing methods for rapid
detection and identification of pathogens of concern. In this sec-
tion, we review some of the newer technologies emerging for the
future and their potential for enhancing methods to detect food-
borne pathogens. As we write this section, however, the reader
should be aware that the technology is rapidly changing, some
recent reviews in relation to the use of biosensors include articles
by Kaittanis et al. (2010), Tallury et al. (2010), and Velusamy
et al. (2010). Two of the overriding factors in sensor detection
methodologies are the ability to obtain a result in a faster time
frame and a preference to automate the system (Anderson and
Taitt 2005). Biosensors are detection devices that use biological
molecules to recognize and quantify analytes of interest—these
analytes include antibodies, receptors, enzymes, nucleic acids,
oligosaccharides, peptides, and so on. A recognition event be-
tween a biomolecule and an agent (e.g., nucleic acid, receptor,
and so on) results in an output that can be measured either opti-
cally, chemically, or electronically. Biosensors should be able to
monitor a sample continuously for analyte and provide a quan-
titative readout (such as measure of concentration).Adenosine Triphosphate DetectionAlthough not a strict biosensor per se, detection of adenosine
triphosphate (ATP) from living cells is used as a means to assess
the quality of cleaning or disinfection of surfaces and utensils.
ATP detection usually involves analysis of a sample swab of an
area; the swab is then combined with a mixture of the enzyme
luciferase and its substrate luciferin. The reaction of luciferin and
luciferase is dependent on the presence of ATP to catalyze the
reaction, which is only found in living cells. The ATP combines
with luciferin to form luciferyl adenylate and pyrophosphate.Luciferin+ATP→Luciferyl+PPiThe luciferyl adenylate reacts with oxygen to form oxy-
luciferin and adenosine monophosphate with the release of
light.Luciferyl adenylate+O 2 →Oxyluciferin+AMP+LightThe enzymatic process results in the release of light, which
can be measured and is proportional to the amount of ATP
present. There are a number of handheld devices that can be
used for this type of testing and are available commercially.
Typical levels of detection are 1–200 CFU. ATP assays are non-
specific and in general provide information of a quality nature;
it cannot detect for specific pathogens or identify the source
of ATP and typically are used as an indicator of hygiene qual-
ity or successful sanitization. In addition, the system may have
errors associated with the sampling technique resulting in false-
positives and false-negatives (Carrick et al. 2001). Modifications
of ATP detection have incorporated the use of fluorescence for