Detection of Salmonella by polymerase chain reaction - Biologia ...

in the presence of 10 6 CFU of E. coli (data not
shown).
Detection of Salmonella in foods
The performance of the developed detection system
was evaluated by the analysis of naturally
contaminated food samples. Two enrichment
protocols were used prior to PCR: a one-step
overnight non-selective enrichment in BPW, and a
three-step selective enrichment comprising a preenrichment
in BPW, two parallel selective enrichments
in Rappaport-Vassiliadis and selenitecystine
media, respectively, and post-enrichment
in LB broth. Of the 18 food samples tested, 9 were
found positive by the ISO method. Identical results
were obtained by the PCR detection coupled
to an overnight enrichment in BPW, but one sample
of chicken meat was found false negative and
two samples of minced meat were found slightly
false positive. PCR after a three-step enrichment
produced identical results as the reference method
(Tab. 4).
Table 4. Detection of Salmonella in foods. Value in
brackets means a slightly positive result (faint band in
the electrophoresis).
No. of
Sample type tested Positive by
samples
PCR-BPW PCR-LB ISO 6579
egg melange 5 2 2 2
minced meat 8 (2) 0 0
chicken meat 2 1 2 2
chicken liver 1 1 1 1
turkey meat 2 2 2 2
Discussion
The aim of the present study was to develop
a rapid PCR-based system for the detection of
Salmonella in foods. We designed original primers
S212/S500 targeted to the fimC gene, which
proved to be Salmonella-specific with no crossreactivity
to the other bacterial species tested.
These primers were also successful in amplifying
a specific DNA product from S. gallinariumpullorum
although type1 fimbriae are not expressed
in this serotype; this result is in agreement
with the observation by Cohen et al.
(1996).
Sensitivity of the developed PCR, 100 CFU
per reaction using a template obtained by a simple
bacterial lysis was similar to that of other comparable
PCR methods recommended for routine
laboratories (Rijpens et al., 1999; Trkov et al.,
1999). Although this might be improved by increasing
the number of amplification cycles, because
of the accompanying danger of false positive
results caused by contamination, a combination of
less amplification cycles with an appropriate enrichment
prior to PCR is preferred.
With PCR-based detection methods, a problem
of false negative results caused by inhibition
may occur. Various components of food, enrichment
media or DNA extraction solutions have
been shown to inhibit PCR (Rossen et al., 1992).
False negative results are more dangerous than the
false positive ones because they may lead to contaminated
samples being judged as safe. To avoid
false negative results, we have prepared an internal
standard, which facilitates monitoring the amplification.
The application of this internal standard
proved to be useful since, with the analysis of some
food samples, inhibition did take place. By repeating
PCR for these samples, eventual false negative
results were avoided.
When testing the complete detection system
with naturally contaminated foods, a good agreement
with the results of the standard method was
observed. However, some discrepancies appeared
at a one-step enrichment used prior to PCR. The
two false positive samples of minced meat, which
produced faint bands in PCR coupled to a one-step
enrichment, but not in the standard method or
when a three-step enrichment was used, were probably
caused by the presence of dead Salmonella
cells in the food. The false negative sample of
chicken meat, which was negative at PCR coupled
to a one-step enrichment, but positive in the standard
method or when a three-step enrichment was
used, was probably contaminated at a low level or
by stressed Salmonella cells. When summarized,
the use of a three-step enrichment prior to PCR
lead to a higher agreement of the results with the
standard method, but required considerably more
time. For screening purposes, a one-step enrichment
may be used but the problem of possible
false positive an false negative results has to be
kept in mind.
Acknowledgements
This study was supported by the Inco-Copernicus
grant (No. PL979044) from EU.
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