726 Part VII: Food Safety
and artificially contaminated cooked ground beef. In
pure culture, the authors used enrichment culturing
for 1 hour, followed by isolation of total RNA from
the pathogen. They performed RT-PCR on the iap,
hlyA,and prfAgenes. Using the iapgene as the tar-
get, the authors were able to detect 10–15 L. mono-
cytogenescells/mL. RT-PCR with the other two
genes was not as sensitive, requiring either a higher
number of organisms or more enrichment time.
When the authors used RT-PCR on the iapgene to
detect the pathogen in ground beef, they detected
organisms at a level of 3 cfu/g of inoculated meat
after 2 hours of enrichment culturing.
DNA Microarrays
Microarrays are composed of a number of discreetly
located DNA probes fixed on a solid substrate such
as glass (Call et al. 2003). Each probe corresponds
to an oligonucleotide specific to a target DNA se-
quence. Call et al. (2003) used probes specific for
unique portions of the 16S rRNA gene in Listeriato
demonstrate how Listeriaspecies could be differen-
tiated by this method. In this procedure, PCR is first
performed using universal primers to amplify all the
16S rRNA genes present in a sample. The various
amplified DNA fragments bind only to the probes
for which they have a complementary sequence. Be-
cause one of the oligonucleotides used in the PCR
contains a fluorescent label, the probes bound to
DNA sequences fluoresce. Pathogens are identified
by the pattern of fluorescing spots in the array. Al-
ternatively, multiple primer sets can be used to
amplify a number of pathogen-specific genes in a
multiplex PCR. Microarrays allow many moreprimer
sets to be used in the PCR step than if detection were
to be performed by gel electrophoresis. This is
because only those products binding to a microarray
probe will be detected, so the researcher is not limit-
ed by the number of resolvable bands on a gel (Call
et al. 2003). Microarrays are relatively cheap and
are able to identify a number of pathogens or
serotypes at once, but they still require culture
enrichment and PCR steps before the results can be
determined.
Nucleic Acid Sequence–Based Amplification
(NASBA)
Beumer and Hazeleger (Beumer and Hazeleger
2003) used in vitro RNA amplification to detect
viable pathogens since RNA is unstable and likely to
be present only in living cells. In nucleic acid
sequence–based amplification (NASBA), total RNA
in a sample is extracted. Messenger RNA is then
used as the target because it predicts viability better
than rRNA or total RNA, and the PCR amplification
step is not used, so no thermocycler is required. In
NASBA, all steps take place at 41°C so that genom-
ic DNA stays in double stranded form and does not
affect the reaction.
During NASBA, three enzymes are required: re-
verse transcriptase, RNaseH, and T7 RNA poly-
merase. Two oligonucleotide primers specific to the
gene of interest are used, and a mixture of both
NTPs and dNTPs are added. The oligonucleotide
complementary to the mRNA binds to its template,
and the reverse transcriptase produces a cDNA mol-
ecule. The RNaseH is then added to digest the RNA
present, at which point the second primer binds to
the cDNA, allowing the reverse transcriptase to
make the complementary strand. This creates a
“minigene” (Cook 2003), which is transcribed into
thousands of RNA transcripts by the T7 RNA poly-
merase. The transcripts can be detected by agarose
gel electrophoresis or by probe hybridization (Cook
2003).
NASBA has been used to detect viable C. jejuni
cells at between 10 and 30 cfu/g of inoculated food
after 48 hours of enrichment (Cook 2003; Uytten-
daele et al. 1999, 1995a). The technique has also
been used to detect viable L. monocytogenesat 10
cfu/60 g meat or seafood product (Blais et al. 1997;
Uyttendaele et al. 1995b), and E. coliat 40 cfu/mL
drinking water (Min and Baeumner 2002). All path-
ogens were detected using 16S rRNA sequences,
and L. monocytogeneswas also detected using the
hlyAmRNA (Blais et al. 1997). The L. monocyto-
genesassay took 3 days to perform, including cul-
ture enrichment (Uyttendaele et al. 1995b).
Aside from ensuring detection of only viable
cells, NASBA removes the need for a costly thermo-
cycler. NASBA still requires that nucleic acids be
extracted from the food samples, so there may be
matrix effects on the enzymes (Cook 2003).PATHOGENSUBTYPING ANDVERIFICATION
METHODSRFLP, RAPD, AFLP, ribotyping, and pulsed-field
gel electrophoresis are molecular-based methods
that are used for further verification after a food