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

crease in salmonellosis in industrialized countries
over the past 35 years has accompanied the centralisation
of food production and processing, despite
of improvements in epidemiological and microbiological
methods. Conventional methods of
Salmonella detection rely on enrichment in nonselective
and selective broths, followed by differential
agar media and serological confirmation.
These methods offer a satisfactory detection limit
and specificity, but require at least 4 or 6 days
to obtain negative or positive results, respectively
(ISO 6579, 1993). There are consequently
efforts to develop new, faster methods. Detection
of Salmonella byPCR,basedontheenzymatic
amplification of a pre-selected region of DNA, is
one such method reducing the time requirement
for the analysis without a negative effect on the
analytical parameters.
Several PCR-based methods for the detection
of Salmonella have been described. These were
based on the identification of various genes, such
as pathogenic determinants invA (Stone et al.,
1994; Burkhalter et al., 1995), spvA (Lampel
et al., 1996), iroB (Baumler et al., 1997), fimbrial
genes fimA (Cohen et al., 1996) and agfA (Doran
et al., 1996), genes encoding for rRNA (Iida
et al., 1993) or Salmonella-specific DNA sequences
with an unknown function (Aabo et al., 1993).
In this work, primers were designed targeted
to the fimC gene, which encodes a chaperone involved
in the synthesis of type 1 fimbriae. These
are proteinaceous filaments localised on the surface
of the bacterial cell and are responsible for
binding to specific receptors on the epithelial cell
during infection. The fim operon is commonly
present in members of the entire family Enterobacteriaceae,
including all Salmonella serotypes (Low
et al., 1996).
An important feature of PCR-based methods
for routine detection of pathogens should be high
reproducibility and reliability. These can be improved
by the application of an internal standard,
which facilitates monitoring the amplification and
helps to recognize false negative results caused by
the inhibition of PCR. For the primers designed,
a mimic internal standard (Lambertz et al., 1998)
was here constructed and its use optimized.
To achieve satisfactory analytical parameters,
food samples have to be appropriately processed
prior to the detection of Salmonella by
PCR. Enrichment in a single non-selective medium
(Manzano et al., 1998), enrichment in a nonselective
medium followed by two parallel selective
media (Cohen et al., 1996), and a successive enrichment
in three media (Aabo et al., 1995) have
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been reported to be used for this purpose. Another
possibility is the use of a combination of nonselective
and selective enrichment with immunomagnetic
separation (Rijpens et al., 1999; Trkov
et al., 1999) In this work, two enrichment protocols
followed by PCR targeted to the fimC gene
were compared with the standard method for the
detection of Salmonella in selected naturally contaminated
foods.
Material and methods
Bacterial strains were from the collection of Food Research
Institute, Bratislava, or they were isolates from
food by the State Veterinary Institute, Bratislava. Bacteria
were cultured on solid or liquid Luria-Bertani
(LB) media (Maniatis et al., 1982). For the determination
of the detection limit, overnight cultures were
serially diluted in LB broth and numbers of viable bacteria
were determined by plating on LB agar.
DNA preparation
Chromosomal DNA was prepared according to Flamm
et al. (1984). Crude lysates were prepared from 1 mL of
the culture grown in the liquid medium, or suspending
one loopful of the culture grown on the solid medium.
Bacterial cells were suspended in 1 mL of 0.8% NaCl,
centrifuged at 8000 g for 10 min, and the pellet was
resuspended in 1% Triton X-100. The suspension was
boiled for 15 min and, after centrifugation the supernatant
was used in PCR.
PCR
Avolumeof2µL of the DNA template solution was
added to 23 µL of the reaction mixture containing
10 mmol/L Tris-HCl, pH 8.8, 50 mmol/L KCl, 1.5
mmol/L MgCl2, 0.05% Tween 20, 200 µmol/L each
dNTP, 250 nmol/L each primer, 1 U Taq DNA polymerase.
Amplification was carried out in a T1 thermal
cycler (Biometra, Göttingen, Germany) with a temperature
programme consisting of the initial denaturation
(1 min at 94 ◦ C), 35 amplification cycles (30 s at 90 ◦ C,
30 s at 54 C, 60 s at 72 ◦ C), and the final extension (8
min at 72 ◦ C). A volume of 10 µL of the PCR product
was analysed by electrophoresis in a 1.5% agarose gel
(Maniatis et al., 1982).
Internal standard preparation
The internal standard was produced by the method described
previously (Drahovská et al., 1999). Briefly,
PCR with E. coli chromosomal DNA as a template
was made with the annealing temperature lowered to
40 ◦ C. From several non-specific products, a fragment
of about 800 bp was selected, extracted from the gel,
and cloned to the pCR2.1 vector (Invitrogen).
Sequencing of the PCR products
Sequencing was performed by an automatic DNA sequencer
(Vistra, Amersham) with individual products
cloned to the pCR2.1 vector.