FLEISCHWIRTSCHAFT international_04_2018

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Fleischwirtschaft international 4_2018
Hygiene
Detection assures food safety
Fig. 3: The analysis of the patterns obtained by PCR can take a number of hours.
and sensitive method for the rapid
enumeration of microorganisms.
The success of the system depends
on the development and use of
suitable staining systems, and
protocols for the separation of
microorganisms from food debris
that would otherwise interfere
with the detection system.
Polymerase chain reaction
Polymerase chain reaction (PCR)
is a method used for the in vitro
enzymatic synthesis of specific
DNA sequences by Taq and other
thermoresistant DNA polymerases.
PCR uses oligonucleotide
primers that are usually
20–30 nucleotides in length and
whose sequence is homologous to
the ends of the genomic DNA
region to be amplified. The
method is performed in repeated
cycles, so that the products of one
cycle serve as the DNA template
for the next cycle, doubling the
number of target DNA copies in
each cycle (HILL, 1996).
Conventional PCR relies on
amplification of the target gene(s)
in a thermo-cycler, separation of
PCR products by gel electrophoresis,
followed by visualization and
analysis of the resulting electrophoretic
patterns (Fig. 3), a
process that can take a number of
hours. The specificity can be subsequently
confirmed by sequencing
the amplified fragment. PCR can
be superior to culture for detecting
the main pathogens in food samples
(ABUBAKAR et al., 2007).
Multiplex PCR (mPCR) combines
several specific primersets
into a single PCR assay for the
simultaneous amplification of
more than one target DNA sequence
(CHAMBERLAIN et al., 1988).
As with conventional or endpoint
PCR, the amplified DNA targets are
separated by agarose gel electrophoresis
and visualized by ethidium
bromide staining. O’REGAN et
al. (2008) developed a real-time
multiplex PCR assay for the detection
of multiple Salmonella
serotypes in chicken samples.
Poultry-associated serotypes detected
in the assay included S.
Enteritidis, S. Gallinarum, S. Typhimurium,
S. Kentucky and S.
Dublin.
RPeal-time PCR allows both the
detection and quantification of a
signal emitted by the amplified
product by using the continuous
measurement of a fluorescent label
during the PCR reaction. The
increase in fluorescence can be
monitored in real time, which
allows accurate quantification over
several orders of magnitude of the
DNA target sequence. Results can
be obtained in an hour or less,
which is considerably faster than
conventional PCR without the he
need for post-amplification steps
such as gel electrophoresis. Realtime
PCR based system is used in
food microbiology, which is a fast
and accurate test for screening food
and environmental samples for
pathogens, e.g. E. coli O157:H7,
Listeria monocytogenes, Enterobacter
sakazakii, Salmonella, Campylobacter
coli etc.
The simplest approach involves
the use of the intercalating fluorescent
dye SYBR Green. This fluorogenic
dye exhibits little fluorescence
when in solution, but emits a
strong fluorescent signal upon
binding to double-stranded DNA.
Thus, as a PCR product accumulates,
fluorescence increases. The
advantages of SYBR Green are that
it is inexpensive, simple, and sensitive.
The disadvantage is that SYBR
Green will bind to any doublestranded
DNA in the reaction,
which may result in an overestimation
of the target concentration. A
second, more accurate and reliable
method is to use fluorescent reporter
probes (TaqMan, Molecular
Beacons, and Scorpions). These
probes depend on Forster Resonance
Energy Transfer (FRET) to
generate the fluorescence signal via
the coupling of a fluorogenic dye
molecule and a quencher moiety to
the same or different oligonucleotide
substrates. The main
advantage of TaqMan probes,
Molecular Beacons and Scorpions
is that they allow for multiplex PCR
assays by using spectrally separated
fluor/quench moieties for each
probe. Multiplex PCR allows internal
controls to be co-amplified and
permits allele discrimination in
single-tube, homogeneous assays.
These hybridization probes afford a
level of discrimination impossible
to obtain with SYBR Green, since
they will only hybridize to true
targets in a PCR and not toprimerdimers
or other spurious products.
However these probes can be expensive
to synthesize, with a separate
probe needed for each target
being analyzed.
Detection of pathogens by PCR
in meats samples often requires
additional evidence of viability
before risks can be assigned. PCR
assay cannot differentiate viable
and non viable organism in the
sample. So amplification of genomic
DNA by PCR has been
shown to be inappropriate for
distinguishing viable from nonviable
bacteria (MASTERS et al.,
1994). Furthermore, in an effort to
address the issue of viability, many
researchers turned to RNA amplification
methods using mRNA as a
target since it is a molecule with a
very short half-life of 0.5 to 2min
due to the rapid degradation by
endogenous RNAases (KING et al.,
1986). For this purpose RT-PCR has
been developed to detect the specific
mRNA. In RT-PCR, an RNA
Fig. 4: Bacteriophages are viruses infecting bacteria and kill them during their multiplication.