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1
Validation of a Diagnostic PCR Method for Routine Analysis of Salmonella spp.
in Animal Feed Samples
Running head: Validation of PCR for Salmonella in feed
Charlotta Lfstrm
a,b
, Charlotta Engdahl Axelsson
b
and Peter Rdstrm
a*
a
Applied Microbiology, Lund Institute of Technology, Lund University, P.O. Box 124,
SE-221 00 Lund,
b
Lantmnnen AnalyCen AB, P.O. Box 905, SE-531 19 Lidkping, Sweden
* Corresponding author. Mailing address: Applied Microbiology, Center for Chemistry and
Chemical Engineering, Lund Institute of Technology, Lund University, P.O. Box 124, SE-221 00
Lund, Sweden. Phone: +46 46 222 3412, Fax: +46 46 222 4203, E-mail:
Peter.Radstrom@tmb.lth.se
Revised manuscript
Click here to download Manuscript: Lofstrom et al revised 27-09-2007.doc
This article is published in: Food Analytical Methods, 2008, 1(1), 23-27
The original publication is available at www.springerlink.com
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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 performed 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
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3
Introduction 1
Food borne diseases such as salmonellosis are recognized as one of the most serious public health 2
concerns today.
1
The problem of salmonellosis related to the food industry is cyclic and animal feed 3
may serve as a reservoir for Salmonella contributing to the spread of the bacteria along the food 4
chain.
2
The conventional culture method used today for detection of Salmonella in feed is laborious 5
and takes 3-7 days to complete.
3
Hence, there is a growing demand for rapid methods for the 6
detection of Salmonella in feed samples. PCR is considered to be one of the most promising 7
techniques to meet this demand and several PCR-based detection methods for Salmonella in food 8
and feed have been developed.
4-8
9
Although the PCR-based methods meet the demands of diagnostic laboratories on detection 10
methods regarding sensitivity, specificity and ease of use, the introduction of the technique for 11
diagnostic use has so far been slow. The technological novelty of the technique, the high investment 12
cost and the lack of officially approved, validated and standardized methods have been mentioned 13
as reasons for this delay.
8, 9
Validation is an important step in the process of standardizing a method, 14
because it provides evidence that the new method gives results at least as good and in agreement 15
with the currently used reference method, as well as proving confirmation of the reproducibility and 16
specificity when used by other laboratories.
8, 9
These data are needed to gain acceptance among 17
authorities and end users of a method, and to speed up the implementation of new rapid PCR-based 18
detection systems in diagnostic laboratories. 19
The aim of this study was to perform a comparative study of a diagnostic PCR procedure
6
and 20
the currently used NMKL reference method
3
for the detection of Salmonella in animal feed 21
samples. The PCR method, based on a simple PCR-compatible enrichment procedure, has been 22
evaluated and found to specifically detect low numbers of viable Salmonella spp. in feed samples 23
without any sample pre-treatment such as DNA extraction or cell lysis prior to PCR. The 24
probability of detecting 1 CFU/25 g feed in the presence of natural background flora was found to 25
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be 0.81.
6
It is therefore of great value to perform a validation study for this method in order to gain 26
acceptance for use on a routine basis. In the first part of this study a comparative study of the PCR 27
and NMKL methods for 250 artificially inoculated or naturally contaminated feed samples of both 28
animal and vegetable origin were performed according to the protocol of NordVal.
10, 11
29
Furthermore, a small interlaboratory study was performed to assess the reproducibility of the PCR 30
method. 31
32
Materials and methods 33
Feed samples. For the comparative study samples of each of the two main categories of feed, i.e. of 34
animal and vegetable origin, as well as other feed related samples, were used (Table 1). For feed of 35
animal origin, 30 samples were not inoculated (not containing salmonella as determined previously 36
by the NMKL method
3
), 14 were inoculated with 1-10 CFU Salmonella/25 g feed, 12 with 10-100 37
CFU/25 g, and 17 were naturally contaminated (unknown salmonella status before analysis). For 38
feed of vegetable origin, 26 samples were not inoculated, 18 were inoculated with 1-10 CFU 39
Salmonella/25 g feed, 16 with 10-100 CFU/25 g and 94 were naturally contaminated. Twenty-three 40
other feed related samples (naturally contaminated) were included in the study (Table 1). For the 41
artificially contaminated samples, half of the samples at each level were inoculated with S. 42
Livingstone and the other half with S. Senftenberg. The inoculation level of S. Senftenberg was 8 43
CFU at the 1-10 CFU level, and 75 CFU at the 10-100 level. The corresponding values for S. 44
Livingstone were 9 CFU at the 1-10 CFU level and 92 CFU at the 10-100 CFU level. 45
46
Salmonella strains. Salmonella enterica ssp. enterica serovar Senftenberg S57 (S. Senftenberg, an 47
animal feed isolate from AnalyCen Nordic AB, Kristianstad, Sweden) and S. Livingstone CCUG 48
39481 (obtained from the Culture Collection, University of Gteborg, Gothenburg, Sweden) were 49
obtained by growth in tryptone soy broth (TSB, Merck, Darmstadt, Germany) at 37C overnight. 50
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The concentration of cells was determined by viable counts on tryptone glucose extract (TGE, 51
Merck) agar plates. The cell suspensions were diluted in saline (0.9% (w/v) NaCl) to concentrations 52
corresponding to 1-10 CFU/ml and 10-100 CFU/ml. 53
54
Sample preparation. Twenty-five grams of each feed was homogenized in 225 ml buffered peptone 55
water (BPW, Lab 46, LabM, Bury, UK) in a sterile plastic bag. The feed homogenates (Table 1) 56
were inoculated with Salmonella and enriched at 37C for 18 h. A small aliquot (0.1 ml) of the 57
samples from this pre-enrichment was analysed further using the NMKL method
3
, including 58
selective enrichment in Rappaport-Vassiliades soy broth (RVS, Oxoid, CM866, Basingstoke, UK) 59
overnight at 42C, plating on selective agar xylose lysine decarboxylase (XLD, Neogen, Acumedia, 60
7166, Lansing, Michigan, USA) and brilliant green agar (BGA, Oxoid, CM329), followed by 61
biochemical and serological identification (see Fig. 1). Samples were withdrawn after the pre- 62
enrichment step for PCR analysis and stored at 20C. Before PCR, samples were thawed and 63
diluted 1:10 in saline and 5 l of the diluted sample was added to the PCR tube. 64
65
PCR conditions. PCR, amplifying a part of the invA gene was run as previously described
6
using a 66
mixture consisting of: 0.2 M of each primer
12
(Scandinavian Gene Synthesis AB, Kping, 67
Sweden), 200 M of each dNTP (Roche Molecular Biochemicals, Mannheim, Germany), 1 PCR 68
buffer (Roche), 0.75 U of Tth DNA polymerase (Roche) and 3 10
4
copies of an internal control 69
DNA fragment.
8
The sample volume used was 5 l and the final volume 25 l. A GeneAmp 9700 70
PCR System thermocycler (Applied Biosystems, Foster City, CA) was used. The temperature 71
program started with a denaturation step of 5 min at 94C, followed by 36 cycles at 94C for 30 s, 72
60C for 30 s and 72C for 40 s, and then 1 cycle at 72C for 7 min. Finally, the samples were 73
cooled to 4C. Samples were analyzed with gel electrophoresis using 1% agarose gels stained with 74
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ethidium bromide, and bands were visualized with the GelDoc 1000 system (Bio-Rad, Hercules, 75
CA) using the Molecular Analyst software (Bio-Rad). 76
77
Data analysis and statistics. After confirmation of the results obtained by PCR, the relative 78
accuracy (AC), relative sensitivity (SE) and relative specificity (SP) were calculated according to 79
the NordVal validation protocol.
10
AC is defined as the degree of correspondence between the 80
response obtained by the alternative method and the reference method on identical samples, as 81
follows: (PA + NA + FP) 100 / (PA + NA + TP + FN + FP), where PA refers to positive 82
agreement, NA to negative agreement, FP to false positives, TP to true positives, and FN to false 83
negatives. SE is defined as the ability of the alternative method to detect the target microorganism 84
compared to the reference method, as follows: (PA + TP) 100 / (PA + FN). SP is defined as the 85
ability of the alternative method not to detect the target microorganism when it is not detected by the 86
reference method, as follows: (NA 100) / (NA + FP). In this study, FP was defined as a negative 87
result for NMKL and a positive result for the PCR method not confirmed by growth; TP was 88
defined as a negative result for NMKL and a positive result for the PCR method confirmed by 89
growth, and FN positive result for NMKL, and a negative result for PCR. 90
To verify that there was no significant difference in the results obtained by the two methods the 91
McNemar test was performed according to Annex F in ISO 16140:2003.
13, 14
Cohens kappa () was 92
calculated as described by NMKL to quantify the degree of agreement between the two methods
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( > 0.80 means very good agreement between methods). 94
95
Results and discussion 96
The comparative trial was conducted in accordance with the guidelines provided by NordVal
10, 11
97
and included the matrix animal feed of both animal and vegetable origin (Table 1). In a comparative 98
trial, parameters such as the relative accuracy, detection level, sensitivity and specificity are 99
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7
evaluated. The relative selectivity in terms of inclusivity and exclusivity of the PCR method have 100
been determined previously.
6
Salmonella strains (n = 101) representing 33 serotypes were correctly 101
identified as Salmonella by both the NMKL and the PCR methods. Strains (n = 43) representing 27 102
bacterial species other than Salmonella were negative according to both methods. Furthermore, a 103
recent study of PCR using the same primer pair showed a 99.6% inclusivity and 100% exclusivity 104
for 364 strains.
8
Several other studies have also confirmed the selectivity of the primers.
12, 16
105
In this study, no significant differences (using the McNemar test) were found between the two 106
methods for a total of 250 artificially or naturally contaminated samples including animal feed of 107
both vegetable and animal origin. Furthermore, a very good agreement between the two methods 108
was obtained using Cohens kappa (Table 1). The relative accuracy, sensitivity and specificity were 109
evaluated for the PCR method in comparison to the standard culture based method currently in use 110
for detection of Salmonella
3
according to the NordVal protocol (Table 1). The relative sensitivity 111
for the matrices animal feed of animal and vegetable origin, as well as when all 249 samples were 112
analysed together were above 95% which is the limit considered acceptable according to NordVal.
10
113
No recommendations concerning the levels for the relative accuracy and relative specificity are 114
given in the standard.
10
To further assess the reproducibility of the PCR method, when performed 115
by different persons in different laboratories, 40 randomly selected artificially contaminated 116
samples were analysed with PCR at two different laboratories. No significant differences were 117
found between the results obtained at the two laboratories (data not shown). 118
When analysing the data in more detail two major trends were noted: (i) the inability to detect 119
Salmonella in acidified feed samples by the NMKL method, and (ii) difficulties in detecting 120
Salmonella in rape seed samples by PCR. The inability of the NMKL method to detect Salmonella 121
in acidified feed samples has been observed previously.
6
One possible reason for this may be that 122
the cells were stressed after acidification and did not recover sufficiently to be able to survive and 123
multiply in the selective RVS broth. Furthermore, it has been shown that Salmonella must reach 124
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levels of 10
4
CFU/ml in RVS broth to enable successful transfer and further growth on the selective 125
agar plates employed in the NMKL method
17
. A total of six false negative results were found in the 126
comparative study. Out of these six, two were rapeseed or rapeseed meal (Table 1). Furthermore, 127
one rapeseed sample totally inhibited the PCR even at 1:100 dilution with no amplification of either 128
the specific product or the internal control. Rapeseed has previously been noted to be PCR- 129
inhibitory
6
with reduced amplification efficiency using the same PCR method. In contrast, 130
Salomonsson et al. (2005) did not have problems with PCR inhibition caused by rape seed samples 131
when detecting salmonella after pre-enrichment in BPW.
5
However, a larger volume was used for 132
the PCR (50 l instead of 25 l in this study) which diluted the inhibitors. Furthermore, the use of 133
another DNA polymerase (rTth instead of Tth) can also explain the differences. The use of alternate 134
DNA polymerases is a convenient way to overcome PCR inhibition and has successfully been 135
applied for different biological matrices.
5, 6, 18
Additionally, Salmonella in one spiked sample 136
(compound feed containing fishmeal and coccidiostatics) was not detected by the PCR method, 137
although the internal control was amplified. As a control, this sample was re-analysed after the 138
validation study was completed using four new 25 g samples inoculated with 4 CFU of Salmonella. 139
All four samples were positive with the PCR method. The other two false negative results for the 140
feed of animal origin were obtained for samples not inoculated with Salmonella (meat meal and fish 141
meal). The samples also proved negative with NMKL when three new 25 g portions were analysed, 142
which indicates that the sample included in the validation study was false positive with the NMKL 143
method, possibly due to cross contamination. 144
Special routines are needed to avoid carry-over contamination during the entire PCR 145
procedure. Particular attention should be paid to the handling of samples to avoid transfer of 146
amplified PCR product to the samples and reaction mixture, as well as to avoid the possibility of 147
contamination of negative wells by adjacent positive wells during gel electrophoresis. One non- 148
spiked sample was false positive with the PCR method. The amplified PCR product showed a faint 149
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band in the gel, which could indicate contamination or the presence of low amounts of Salmonella 150
in the sample. Reasons for this result might be carry-over contamination during preparation of the 151
samples or contamination from adjacent wells during gel electrophoresis. The sample was 152
reanalysed using both the NMKL and PCR methods with negative results. However, there is a 153
possibility that the sample might have been contaminated with Salmonella at a low level, which was 154
not detectable. The issue of weak bands should therefore be considered in the final protocol for the 155
alternative method. To circumvent these problems and further speed up the analysis, real-time PCR 156
or PCR-ELISA might be used instead of electrophoresis to detect the amplicon. Several recent 157
studies have successfully detected the same amplicon using PCR followed by ELISA
19
and real- 158
time PCR using SYBRGreen
19, 20
or TaqMan probes
19-21
for detection. 159
In conclusion, the PCR method validated in this study enabled the detection of low numbers 160
of Salmonella in less than 24 h, compared to at least 3 days using the NMKL method (Fig. 1). For 161
the problematic sample type rapeseed further studies are needed to achieve better pre-PCR 162
processing to overcome the inhibitory effect. For acidified soy samples more positives were found 163
with the PCR method than with the NMKL method, indicating difficulties in the recovery of 164
sublethally damaged Salmonella cells by selective enrichment. To obtain NordVal approval the 165
study needs to be supplemented with an additional interlaboratory study. However, the specificity, 166
simplicity and speed of the PCR method makes it suitable for routine analysis of large numbers of 167
samples and the implementation of the method in industry will help improve safety in the food 168
production chain. 169
170
Acknowledgements 171
The authors wish to thank Dr. Halfdan Grage for help with the statistical analysis and Desire 172
Andersson for technical assistance. This work was financially supported by the Swedish Agency for 173
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Innovation Systems (VINNOVA), and the Foundation of the Swedish Farmers' Supply and 174
Crop Marketing Cooperation (SL-stiftelsen). 175
176
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References 177
178
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Oslo, Norway 2007). 201
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Table 1 Comparison of the results in the comparative trial obtained by PCR and the reference culture method
a
. 211
Category Sample type PA NA FN TP FP Total
b
AC
(%)
SE
(%)
SP
(%)

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 meal and coccidiostatics) 5 4 1 0 0 10 90.0 83.3 100.0
Animal total 25 43 3 2 0 73 93.2 96.4 100.0 0.85
Vegetable origin Ingredients, not heat treated (cereals, pulses and rape seed) 7 18 1 1 0 27 92.6 100.0 100.0
Ingredients, acidified (soybean meal, rape seed meal) 7 48 0 1 1 57 98.2 114.3 98.0
Compound feed (pellets) 8 9 0 0 0 17 100.0 100.0 100.0
Ingredients, heat treated (soybean meal, rapeseed meal, palm
kernel expeller)
14 36 1 0 1 52 98.1 93.3 97.3
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
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32
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41
42
43
44
45
46
47
48
49
14
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, 212
FP: False Positive, AC: Relative Accuracy, SE: Relative Sensitivity, SP: Relative Specificity, N = PA +NA + FN + TP + FP 213
b
The PCR for one rape seed sample was totally inhibited (no specific product or internal control band produced) even at 1:100 dilution. 214
This result is considered inconclusive and was not included in the analysis. The total number of samples included in the statistical analysis was 215
therefore 249. 216
c
-, calculation of SE was not possible because PA + FN = 0 217
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FIGURE LEGEND 218
Fig. 1. Overview of the validation study set-up. 219
220
25 g feed + 225 ml BPW
Enrichment
Biochemical and serological
identification
Dilution in saline PCR (invA)
37C, 18 h
Enrichment
(RVS)
42C, 24 h
Selective plating (XLD, BGA) 37C, 24 h
Day 0
Day 1
Day 2
Day 3
Figure 1
Click here to download Figure: Lofstrom et al Fig 1.ppt