Food Anal.
Methods (2008) 1:23–27
DOI 10.1007/s12161-007-9003-2
Validation of a Diagnostic PCR Method for Routine Analysis
of Salmonella spp. in Animal Feed Samples
Charlotta Löfström & Charlotta Engdahl Axelsson &
Peter Rådström
Received: 20 August 2007 / Accepted: 28 September 2007 / Published online: 29 December 2007
# Springer Science + Business Media, LLC 2007
Abstract As a part of a validation study, a comparative
study of a PCR method and the standard culture-based
method NMKL-71, for detection of Salmonella, was per-
formed according to the validation protocol from the
Nordic validation organ for validation of alternative
microbiological methods (NordVal) on 250 artificially or
naturally contaminated animal feed samples. The PCR
method is based on culture enrichment in buffered peptone
water followed by PCR using the DNA polymerase Tth and
an internal amplification control. No significant difference
was found between the two methods. The relative accuracy,
relative sensitivity and relative specificity were found to be
96.0, 97.3, and 98.8%, respectively. PCR inhibition was
observed for rape seed samples. For the acidified feed
samples, more Salmonella-positive samples were found
with the PCR method compared to the NMKL method.
This study focuses on the growing demand for validated
diagnostic PCR methods for routine analysis of animal feed
and food samples to assure safety in the food production
chain.
Keywords Animal Feed . NordVal . Salmonella .
Validation . PCR . Polymerase Chain Reaction
C. Löfström : P. Rådström (*)
Applied Microbiology, Lund Institute of Technology,
Lund University,
P.O. Box 124, SE-221 00 Lund, Sweden
e-mail: Peter.Radstrom@tmb.lth.se
C. Löfström : C. E. Axelsson
Lantmännen AnalyCen AB,
P.O. Box 905, SE-531 19 Lidköping, Sweden
Introduction
Food-borne diseases such as salmonellosis are recognized
as one of the most serious public health concerns today
(Tirado and Schmidt 2001). The problem of salmonellosis
related to the food industry is cyclic, and animal feed
may serve as a reservoir for Salmonella contributing to the
spread of the bacteria along the food chain (Davies and
Hinton 2000). The conventional culture method used today
for detection of Salmonella in feed is laborious and takes
3–7 days to complete (Anonymous 1999). Hence, there is a
growing demand for rapid methods for the detection of
Salmonella in feed samples. Polymerase chain reaction
(PCR) is considered to be one of the most promising
techniques to meet this demand, and several PCR-based
detection methods for Salmonella in food and feed have
been developed (Hoorfar et al. 2000; Salomonsson et al.
2005; Löfström et al. 2004; Malorny et al. 2003a, 2004).
Although the PCR-based methods meet the demands of
diagnostic laboratories on detection methods regarding
sensitivity, specificity, and ease of use, the introduction of
the technique for diagnostic use has so far been slow. The
technological novelty of the technique, the high investment
cost, and the lack of officially approved, validated, and
standardized methods have been mentioned as reasons
for this delay (Malorny et al. 2003a, b). Validation is an
important step in the process of standardizing a method
because it provides evidence that the new method gives
results at least as good and in agreement with the currently
used reference method, as well as proving confirmation
of the reproducibility and specificity when used by other
laboratories (Malorny et al. 2003a, b). These data are needed
to gain acceptance among authorities and end users of a
method and to speed up the implementation of new rapid
PCR-based detection systems in diagnostic laboratories.
24 Food Anal. Methods (2008) 1:23–27

The aim of this study was to perform a comparative study
of a diagnostic PCR procedure (Löfström et al. 2004) and the
currently used NMKL reference method (Anonymous
1999) for the detection of Salmonella in animal feed sam-
ples. The PCR method, based on a simple PCR-compatible
enrichment procedure, has been evaluated and found to
specifically detect low numbers of viable Salmonella spp.
in feed samples without any sample pretreatment such as
DNA extraction or cell lysis before PCR. The probability of
detecting 1 CFU/25 g feed in the presence of natural
background flora was found to be 0.81 (Löfström et al.
2004). It is therefore of great value to perform a validation
study for this method to gain acceptance for use on a
routine basis. In the first part of this study, a comparative
study of the PCR and NMKL methods for 250 artificially
inoculated or naturally contaminated feed samples of both
animal and vegetable origin were performed according to
the protocol of NordVal (Anonymous 2002; Qvist 2007).
Furthermore, a small interlaboratory study was performed
to assess the reproducibility of the PCR method.
Materials and Methods
Feed Samples For the comparative study samples of each
of the two main categories of feed, i.e., of animal and
vegetable origin, as well as other feed-related samples, were
used (Table 1). For feed of animal origin, 30 samples were
not inoculated (not containing salmonella as determined
previously by the NMKL method (Anonymous 1999), 14
were inoculated with 1–10 CFU Salmonella/25 g feed, 12
with 10–100 CFU/25 g, and 17 were naturally contami-
nated (unknown salmonella status before analysis). For feed
of vegetable origin, 26 samples were not inoculated, 18
were inoculated with 1–10 CFU Salmonella/25 g feed, 16
with 10–100 CFU/25 g, and 94 were naturally contami-
nated. Twenty-three other feed-related samples (naturally
contaminated) were included in the study (Table 1). For the
artificially contaminated samples, half of the samples at
each level were inoculated with S. Livingstone and the
other half with S. Senftenberg. The inoculation level of
S. Senftenberg was 8 CFU at the 1–10 CFU level, and 75
CFU at the 10–100 level. The corresponding values for
S. Livingstone were 9 CFU at the 1–10 CFU level and 92
CFU at the 10–100 CFU level.
Salmonella Strains Salmonella enterica ssp. enterica serovar
Senftenberg S57 (S. Senftenberg, an animal feed isolate from
AnalyCen Nordic AB, Kristianstad, Sweden) and S. Living-
stone CCUG 39481 (obtained from the Culture Collection,
University of Göteborg, Gothenburg, Sweden) were obtained
by growth in tryptone soy broth (TSB, Merck, Darmstadt,

Table 1 Comparison of the results in the comparative trial obtained by PCR and the reference culture methoda

Category Sample type PA NA FN TP FP Totalb AC (%) SE (%) SP (%) κ

Animal origin Fishmeal 8 12 1 0 0 21 95.2 88.9 100.0

Meat meal and bone meal 8 21 1 2 0 32 90.6 111.1 100.0
Compound feed (pet food pellets) 4 6 0 0 0 10 100.0 100.0 100.0
Compound feed (containing fish 5 4 1 0 0 10 90.0 83.3 100.0
meal and coccidiostatics)
Animal total 25 43 3 2 0 73 93.2 96.4 100.0 0.85
Vegetable origin Ingredients, not heat treated 7 18 1 1 0 27 92.6 100.0 100.0
(cereals, pulses and rape seed)
Ingredients, acidified 7 48 0 1 1 57 98.2 114.3 98.0
(soybean meal, rape seed meal)
Compound feed (pellets) 8 9 0 0 0 17 100.0 100.0 100.0
Ingredients, heat treated (soybean meal, 14 36 1 0 1 52 98.1 93.3 97.3
rapeseed meal, palm kernel expeller)
Vegetable total 36 111 2 2 2 153 97.4 100.0 98.2 0.90
Other Environmental 0 3 0 0 0 3 100.0 –c 100.0
Feed concentrate 7 12 1 0 0 20 95.0 87.5 100.0
Other total 7 15 1 0 0 23 95.7 87.5 100.0 0.90
Total 68 169 6 4 2 249 96.0 97.3 98.8 0.88
a
PA, positive agreement; NA, negative agreement; TP, true positive; FN, false negative;
FP, false positive; AC, relative accuracy; SE, relative sensitivity; SP, relative specificity; N ¼ PA þ NA þ FN þ TP þ FP
b
The PCR for one rape seed sample was totally inhibited (no specific product or internal control band produced) even at 1:100 dilution. This
result is considered inconclusive and was not included in the analysis. The total number of samples included in the statistical analysis was
therefore 249.
c
calculation of SE was not possible because PA þ FN ¼ 0
Food Anal. Methods (2008) 1:23–27
Germany) at 37°C overnight. The concentration of cells was
determined by viable counts on tryptone glucose extract (TGE,
Merck) agar plates. The cell suspensions were diluted in
saline [0.9% (w/v) NaCl] to concentrations corresponding to
1–10 CFU/ml and 10–100 CFU/ml.
Sample Preparation Twenty-five grams of each feed was
homogenized in 225 ml buffered peptone water (BPW, Lab
46, LabM, Bury, UK) in a sterile plastic bag. The feed
homogenates (Table 1) were inoculated with Salmonella
and enriched at 37°C for 18 h. A small aliquot (0.1 ml) of
the samples from this pre-enrichment was analyzed further
using the NMKL method (Anonymous 1999), including
selective enrichment in Rappaport-Vassiliades soy broth
(RVS, Oxoid, CM866, Basingstoke, UK) overnight at 42°C,
plating on selective agar xylose lysine decarboxylase (XLD,
Neogen, Acumedia, 7166, Lansing, Michigan, USA) and
brilliant green agar (BGA, Oxoid, CM329), followed by
biochemical and serological identification (see Fig. 1). The
samples were withdrawn after the pre-enrichment step for
PCR analysis and stored at −20°C. Before PCR, the
samples were thawed and diluted 1:10 in saline, and 5 μl
of the diluted sample was added to the PCR tube.
PCR Conditions PCR, amplifying a part of the invA gene
was run as previously described (Löfström et al. 2004)
using a mixture consisting of: 0.2 μM of each primer (Rahn
et al. 1992; Scandinavian Gene Synthesis AB, Köping,
Sweden), 200 μM of each dNTP (Roche Molecular Bio-
chemicals, Mannheim, Germany), 1 × PCR buffer (Roche),
0.75 U of Tth DNA polymerase (Roche), and 3×104
copies of an internal control DNA fragment (Malorny
et al. 2003a). The sample volume used was 5 μl and the
final volume 25 μl. A GeneAmp 9700 PCR System ther-
mocycler (Applied Biosystems, Foster City, CA, USA)
Fig. 1 Overview of the
validation study set-up Day 0
Day 1
Day 2 Selective plating (XLD, BGA)
Day 3 Biochemical and serological
identification
25
was used. The temperature program started with a dena-
turation step of 5 min at 94°C, followed by 36 cycles at
94°C for 30 s, 60°C for 30 s, and 72°C for 40 s, and then 1
cycle at 72°C for 7 min. Finally, the samples were cooled
to 4°C. The samples were analyzed with gel electro-
phoresis using 1% agarose gels stained with ethidium
bromide, and bands were visualized with the GelDoc 1000
system (Bio-Rad, Hercules, CA, USA) using the Molec-
ular Analyst software (Bio-Rad).
Data Analysis and Statistics After confirmation of the
results obtained by PCR, the relative accuracy (AC), rela-
tive sensitivity (SE), and relative specificity (SP) were
calculated according to the NordVal validation protocol
(Anonymous 2002). AC is defined as the degree of corre-
spondence between the response obtained by the alterna-
tive method and the reference method on identical samples,
as follows: (PA + NA + FP) × 100/(PA + NA + TP + FN +
FP), where PA refers to positive agreement, NA to negative
agreement, FP to false positives, TP to true positives, and
FN to false negatives. SE is defined as the ability of the
alternative method to detect the target microorganism
compared to the reference method, as follows: (PA + TP) ×
100/(PA + FN). SP is defined as the ability of the alternative
method not to detect the target microorganism when it is not
detected by the reference method, as follows: (NA × 100)/
(NA + FP). In this study, FP was defined as a negative result
for NMKL and a positive result for the PCR method not
confirmed by growth; TP was defined as a negative result for
NMKL and a positive result for the PCR method confirmed
by growth, and FN positive result for NMKL, and a negative
result for PCR.
To verify that there was no significant difference in the
results obtained by the two methods, the McNemar test
was performed according to Annex F in ISO 16140:2003
25 g feed + 225 ml BPW
Enrichment 37°C, 18 h
Enrichment 42°C, 24 h Dilution in saline PCR (invA)
(RVS)
37°C, 24 h
26
(Agresti 1996; Anonymous 2003). Cohen’s kappa (κ) was
calculated as described by NMKL to quantify the degree
of agreement between the two methods (Anonymous 2007;
κ>0.80 means very good agreement between methods).
Results and Discussion
The comparative trial was conducted in accordance with
the guidelines provided by NordVal (Anonymous 2002;
Qvist 2007) and included the matrix animal feed of both
animal and vegetable origin (Table 1). In a comparative
trial, parameters such as the relative accuracy, detection
level, sensitivity, and specificity are evaluated. The relative
selectivity in terms of inclusivity and exclusivity of the
PCR method have been determined previously (Löfström
et al. 2004). Salmonella strains (n=101) representing 33
serotypes were correctly identified as Salmonella by both
the NMKL and the PCR methods. Strains (n=43) repre-
senting 27 bacterial species other than Salmonella were
negative according to both methods. Furthermore, a recent
study of PCR using the same primer pair showed a 99.6%
inclusivity and 100% exclusivity for 364 strains (Malorny
et al. 2003a). Several other studies have also confirmed
the selectivity of the primers (Rahn et al. 1992; Chen
et al. 1997).
In this study, no significant differences (using the
McNemar test) were found between the two methods for
a total of 250 artificially or naturally contaminated sam-
ples including animal feed of both vegetable and animal
origin. Furthermore, a very good agreement between the
two methods was obtained using Cohens kappa (Table 1).
The relative accuracy, sensitivity, and specificity were eval-
uated for the PCR method in comparison to the standard
culture based method currently in use for detection of
Salmonella (Anonymous 1999) according to the NordVal
protocol (Table 1). The relative sensitivity for the matrices
animal feed of animal and vegetable origin, as well as
when all 249 samples were analyzed together were above
95%, which is the limit considered acceptable according
to NordVal (Anonymous 2002). No recommendations
concerning the levels for the relative accuracy and relative
specificity are given in the standard (Anonymous 2002).
To further assess the reproducibility of the PCR method,
when performed by different persons in different labora-
tories, 40 randomly selected artificially contaminated sam-
ples were analyzed with PCR at two different laboratories.
No significant differences were found between the results
obtained at the two laboratories (data not shown).
When analyzing the data in more detail, two major
trends were noted: (1) the inability to detect Salmonella in
acidified feed samples by the NMKL method and (2)
Food Anal. Methods (2008) 1:23–27
difficulties in detecting Salmonella in rape seed samples
by PCR. The inability of the NMKL method to detect
Salmonella in acidified feed samples has been observed
previously (Löfström et al. 2004). One possible reason for
this may be that the cells were stressed after acidification
and did not recover sufficiently to be able to survive
and multiply in the selective RVS broth. Furthermore, it
has been shown that Salmonella must reach levels of
104 CFU/ml in RVS broth to enable successful transfer
and further growth on the selective agar plates employed
in the NMKL method (Beckers et al. 1987). A total of
six false negative results were found in the comparative
study. Out of these six, two were rapeseed or rapeseed
meal (Table 1). Furthermore, one rapeseed sample totally
inhibited the PCR even at 1:100 dilution with no ampli-
fication of either the specific product or the internal control.
Rapeseed has previously been noted to be PCR-inhibitory
(Löfström et al. 2004) with reduced amplification efficien-
cy using the same PCR method. In contrast, Salomonsson
et al. (2005) did not have problems with PCR inhibition
caused by rapeseed samples when detecting salmonella
after pre-enrichment in BPW (Salomonsson et al. 2005).
However, a larger volume was used for the PCR (50 μl
instead of 25 μl in this study), which diluted the inhibitors.
Furthermore, the use of another DNA polymerase (rTth
instead of Tth) can also explain the differences. The use of
alternate DNA polymerases is a convenient way to over-
come PCR inhibition and has successfully been applied for
different biological matrices (Salomonsson et al. 2005;
Löfström et al. 2004; Abu Al-Soud and Rådström 1998).
Additionally, Salmonella in one spiked sample (compound
feed containing fishmeal and coccidiostatics) was not
detected by the PCR method, although the internal control
was amplified. As a control, this sample was reanalyzed
after the validation study was completed using four new
25-g samples inoculated with 4 CFU of Salmonella. All
four samples were positive with the PCR method. The
other two false negative results for the feed of animal
origin were obtained for samples not inoculated with
Salmonella (meat meal and fish meal). The samples also
proved negative with NMKL when three new 25 g por-
tions were analyzed, which indicates that the sample
included in the validation study was false positive with
the NMKL method, possibly due to cross contamination.
Special routines are needed to avoid carry-over contam-
ination during the entire PCR procedure. Particular atten-
tion should be paid to the handling of samples to avoid
transfer of amplified PCR product to the samples and
reaction mixture, as well as to avoid the possibility of
contamination of negative wells by adjacent positive wells
during gel electrophoresis. One non-spiked sample was
false positive with the PCR method. The amplified PCR
Food Anal. Methods (2008) 1:23–27
product showed a faint band in the gel, which could indi-
cate contamination or the presence of low amounts of
Salmonella in the sample. Reasons for this result might be
carry-over contamination during preparation of the samples
or contamination from adjacent wells during gel electro-
phoresis. The sample was reanalyzed using both the NMKL
and PCR methods with negative results. However, there is a
possibility that the sample might have been contaminated
with Salmonella at a low level, which was not detectable.
The issue of weak bands should therefore be considered in
the final protocol for the alternative method. To circumvent
these problems and further speed up the analysis, real-time
PCR or PCR-ELISA might be used instead of electro-
phoresis to detect the amplicon. Several recent studies have
successfully detected the same amplicon using PCR fol-
lowed by ELISA (Perelle et al. 2004) and real-time PCR
using SYBRGreen (Perelle et al. 2004; Wolffs et al. 2006)
or TaqMan probes (Perelle et al. 2004; Wolffs et al. 2006;
Hein et al. 2006) for detection.
In conclusion, the PCR method validated in this study
enabled the detection of low numbers of Salmonella in less
than 24 h, compared to at least 3 days using the NMKL
method (Fig. 1). For the problematic sample type rapeseed,
further studies are needed to achieve better pre-PCR pro-
cessing to overcome the inhibitory effect. For acidified soy
samples, more positives were found with the PCR method
than with the NMKL method, indicating difficulties in the
recovery of sublethally damaged Salmonella cells by
selective enrichment. To obtain NordVal approval the study
needs to be supplemented with an additional interlaboratory
study. However, the specificity, simplicity, and speed of the
PCR method makes it suitable for routine analysis of large
numbers of samples and the implementation of the method
in industry will help improve safety in the food production
chain.
Acknowledgement The authors wish to thank Dr. Halfdan Grage
for help with the statistical analysis and Desirée Andersson for
27
technical assistance. This work was financially supported by the
Swedish Agency for Innovation Systems (VINNOVA), and the
Foundation of the Swedish Farmers’ Supply and Crop Marketing
Cooperation (SL-stiftelsen).
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