Löfström Et Al 2008 Pre-print FANM 1(1), 23-37

8/12/2019 Lfstrm Et Al 2008 Pre-print FANM 1(1), 23-37

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1

Validation of a Diagnostic PCR Method for Routine Analysis ofSalmonella spp.

in Animal Feed Samples

Running head: Validation of PCR for Salmonellain feed

Charlotta Lfstrma,b, Charlotta Engdahl Axelssonband Peter Rdstrma*

aApplied Microbiology, Lund Institute of Technology, Lund University, P.O. Box 124,

SE-221 00 Lund, bLantmnnen 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:

[email protected]

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 Tthand 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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Introduction1

Food borne diseases such as salmonellosis are recognized as one of the most serious public health2

concerns today.1The problem of salmonellosis related to the food industry is cyclic and animal feed3

may serve as a reservoir for Salmonella contributing to the spread of the bacteria along the food4

chain.2The conventional culture method used today for detection of Salmonella in feed is laborious5

and takes 3-7 days to complete.3 Hence, there is a growing demand for rapid methods for the6

detection of Salmonella in feed samples. PCR is considered to be one of the most promising7

techniques to meet this demand and several PCR-based detection methods for Salmonella in food8

and feed have been developed.4-89

Although the PCR-based methods meet the demands of diagnostic laboratories on detection10

methods regarding sensitivity, specificity and ease of use, the introduction of the technique for11

diagnostic use has so far been slow. The technological novelty of the technique, the high investment12

cost and the lack of officially approved, validated and standardized methods have been mentioned13

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 agreement15

with the currently used reference method, as well as proving confirmation of the reproducibility and16

specificity when used by other laboratories.8, 9These data are needed to gain acceptance among17

authorities and end users of a method, and to speed up the implementation of new rapid PCR-based18

detection systems in diagnostic laboratories.19

The aim of this study was to perform a comparative study of a diagnostic PCR procedure6and20

the currently used NMKL reference method3 for the detection of Salmonella in animal feed21

samples. The PCR method, based on a simple PCR-compatible enrichment procedure, has been22

evaluated and found to specifically detect low numbers of viable Salmonella spp. in feed samples23

without any sample pre-treatment such as DNA extraction or cell lysis prior to PCR. The24

probability of detecting 1 CFU/25 g feed in the presence of natural background flora was found to25


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be 0.81.6It is therefore of great value to perform a validation study for this method in order to gain26

acceptance for use on a routine basis. In the first part of this study a comparative study of the PCR27

and NMKL methods for 250 artificially inoculated or naturally contaminated feed samples of both28

animal and vegetable origin were performed according to the protocol of NordVal.10, 11

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Furthermore, a small interlaboratory study was performed to assess the reproducibility of the PCR30

method.31

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Materials and methods33

Feed samples.For the comparative study samples of each of the two main categories of feed, i.e. of34

animal and vegetable origin, as well as other feed related samples, were used (Table 1). For feed of35

animal origin, 30 samples were not inoculated (not containing salmonella as determined previously36

by the NMKL method3), 14 were inoculated with 1-10 CFU Salmonella/25 g feed, 12 with 10-10037

CFU/25 g, and 17 were naturally contaminated (unknown salmonella status before analysis). For38

feed of vegetable origin, 26 samples were not inoculated, 18 were inoculated with 1-10 CFU39

Salmonella/25 g feed, 16 with 10-100 CFU/25 g and 94 were naturally contaminated. Twenty-three40

other feed related samples (naturally contaminated) were included in the study (Table 1). For the41

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 843

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, an47

animal feed isolate from AnalyCen Nordic AB, Kristianstad, Sweden) and S. Livingstone CCUG48

39481 (obtained from the Culture Collection, University of Gteborg, Gothenburg, Sweden) were49

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 concentrations52

corresponding to 1-10 CFU/ml and 10-100 CFU/ml.53

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Sample preparation.Twenty-five grams of each feed was homogenized in 225 ml buffered peptone55

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 the57

samples from this pre-enrichment was analysed further using the NMKL method3, including58

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 by61

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 and63

diluted 1:10 in saline and 5 l of the diluted sample was added to the PCR tube.64

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PCR conditions. PCR, amplifying a part of the invA gene was run as previously described6using a66

mixture consisting of: 0.2 M of each primer12 (Scandinavian Gene Synthesis AB, Kping,67

Sweden), 200 M of each dNTP (Roche Molecular Biochemicals, Mannheim, Germany), 1 PCR68

buffer (Roche), 0.75 U of TthDNA polymerase (Roche) and 3 104copies of an internal control69

DNA fragment.8The sample volume used was 5 l and the final volume 25 l. A GeneAmp 970070

PCR System thermocycler (Applied Biosystems, Foster City, CA) was used. The temperature71

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 were73

cooled to 4C. Samples were analyzed with gel electrophoresis using 1% agarose gels stained with74


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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

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Data analysis and statistics. After confirmation of the results obtained by PCR, the relative78

accuracy (AC), relative sensitivity (SE) and relative specificity (SP) were calculated according to79

the NordVal validation protocol.10 AC is defined as the degree of correspondence between the80

response obtained by the alternative method and the reference method on identical samples, as81

follows: (PA + NA + FP) 100 / (PA + NA + TP + FN + FP), where PA refers to positive82

agreement, NA to negative agreement, FP to false positives, TP to true positives, and FN to false83

negatives. SE is defined as the ability of the alternative method to detect the target microorganism84

compared to the reference method, as follows: (PA + TP) 100 / (PA + FN). SP is defined as the85

ability of the alternative methodnot to detect the target microorganism when it is not detected by the86

reference method, as follows: (NA 100) / (NA + FP). In this study, FP was defined as a negative87

result for NMKL and a positive result for the PCR method not confirmed by growth; TP was88

defined as a negative result for NMKL and a positive result for the PCR method confirmed by89

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 the91

McNemar test was performed according to Annex F in ISO 16140:2003.13, 14Cohens kappa () was92

calculated as described by NMKL to quantify the degree of agreement between the two methods 1593

( > 0.80 means very good agreement between methods).94

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Results and discussion96

The comparative trial was conducted in accordance with the guidelines provided by NordVal10, 1197

and included the matrix animal feed of both animal and vegetable origin (Table 1). In a comparative98

trial, parameters such as the relative accuracy, detection level, sensitivity and specificity are99


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evaluated. The relative selectivity in terms of inclusivity and exclusivity of the PCR method have100

been determined previously.6 Salmonella strains (n = 101) representing 33 serotypes were correctly101

identified as Salmonellaby both the NMKL and the PCR methods. Strains (n = 43) representing 27102

bacterial species other than Salmonellawere negative according to both methods. Furthermore, a103

recent study of PCR using the same primer pair showed a 99.6% inclusivity and 100% exclusivity104

for 364 strains.8Several other studies have also confirmed the selectivity of the primers.12, 16105

In this study, no significant differences (using the McNemar test) were found between the two106

methods for a total of 250 artificially or naturally contaminated samples including animal feed of107

both vegetable and animal origin. Furthermore, a very good agreement between the two methods108

was obtained using Cohens kappa (Table 1). The relative accuracy, sensitivity and specificity were109

evaluated for the PCR method in comparison to the standard culture based method currently in use110

for detection of Salmonella3according to the NordVal protocol (Table 1). The relative sensitivity111

for the matrices animal feed of animal and vegetable origin, as well as when all 249 samples were112

analysed together were above 95% which is the limit considered acceptable according to NordVal.10113

No recommendations concerning the levels for the relative accuracy and relative specificity are114

given in the standard.10To further assess the reproducibility of the PCR method, when performed115

by different persons in different laboratories, 40 randomly selected artificially contaminated116

samples were analysed with PCR at two different laboratories. No significant differences were117

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 detect119

Salmonella in acidified feed samples by the NMKL method, and (ii) difficulties in detecting120

Salmonellain rape seed samples by PCR. The inability of the NMKL method to detect Salmonella121

in acidified feed samples has been observed previously.6One possible reason for this may be that122

the cells were stressed after acidification and did not recover sufficiently to be able to survive and123

multiply in the selective RVS broth. Furthermore, it has been shown that Salmonellamust reach124


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levels of 104CFU/ml in RVS broth to enable successful transfer and further growth on the selective125

agar plates employed in the NMKL method17. A total of six false negative results were found in the126

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 either128

the specific product or the internal control. Rapeseed has previously been noted to be PCR-129

inhibitory6 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 samples131

when detecting salmonella after pre-enrichment in BPW.5However, a larger volume was used for132

the PCR (50 l instead of 25 l in this study) which diluted the inhibitors. Furthermore, the use of133

another DNA polymerase (rTthinstead of Tth) can also explain the differences. The use of alternate134

DNA polymerases is a convenient way to overcome PCR inhibition and has successfully been135

applied for different biological matrices.5, 6, 18 Additionally, Salmonella in one spiked sample136

(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 the138

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 the140

feed of animal origin were obtained for samples not inoculated with Salmonella(meat meal and fish141

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 NMKL143

method, possibly due to cross contamination.144

Special routines are needed to avoid carry-over contamination during the entire PCR145

procedure. Particular attention should be paid to the handling of samples to avoid transfer of146

amplified PCR product to the samples and reaction mixture, as well as to avoid the possibility of147

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 faint149


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band in the gel, which could indicate contamination or the presence of low amounts of Salmonella150

in the sample. Reasons for this result might be carry-over contamination during preparation of the151

samples or contamination from adjacent wells during gel electrophoresis. The sample was152

reanalysed using both the NMKL and PCR methods with negative results. However, there is a153

possibility that the sample might have been contaminated with Salmonellaat a low level, which was154

not detectable. The issue of weak bands should therefore be considered in the final protocol for the155

alternative method. To circumvent these problems and further speed up the analysis, real-time PCR156

or PCR-ELISA might be used instead of electrophoresis to detect the amplicon. Several recent157

studies have successfully detected the same amplicon using PCR followed by ELISA19 and real-158

time PCR using SYBRGreen19, 20or TaqMan probes19-21for detection.159

In conclusion, the PCR method validated in this study enabled the detection of low numbers160

of Salmonellain less than 24 h, compared to at least 3 days using the NMKL method (Fig. 1). For161

the problematic sample type rapeseed further studies are needed to achieve better pre-PCR162

processing to overcome the inhibitory effect. For acidified soy samples more positives were found163

with the PCR method than with the NMKL method, indicating difficulties in the recovery of164

sublethally damaged Salmonella cells by selective enrichment. To obtain NordVal approval the165

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 of167

samples and the implementation of the method in industry will help improve safety in the food168

production chain.169

170

Acknowledgements171

The authors wish to thank Dr. Halfdan Grage for help with the statistical analysis and Desire172

Andersson for technical assistance. This work was financially supported by the Swedish Agency for173


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Innovation Systems (VINNOVA), and the Foundation of the Swedish Farmers' Supply and174

Crop Marketing Cooperation (SL-stiftelsen).175

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References177

178

1. C. Tirado and K. Schmidt, J Infect 43, 80 (2001).179

2. R.H. Davies and M.H. Hinton, In: Salmonella in Domestic Animals, edited by C. Wray and A.180

Wray (Cabi Publishing, Wallingford, UK, 2000), p. 285.181

3. Anonymous,Method no 71, 5th ed (Nordic Committee on Food Analysis (NMKL), bo,182

Finland 1999).183

4. J. Hoorfar, P. Ahrens and P. Rdstrm, J Clin Microbiol 38, 3429 (2000).184

5. A.C. Salomonsson, A. Aspn, S. Johansson, A. Heino and P. Hggblom, J Rapid Methods Auto185

Microbiol 13, 96 (2005).186

6. C. Lfstrm, R. Knutsson, C. Engdahl Axelsson and P. Rdstrm, Appl Environ Microbiol 70,187

69 (2004).188

7. B. Malorny, E. Paccassoni, P. Fach, C. Bunge, A. Martin and R. Helmuth, Appl Environ189

Microbiol 70, 7046 (2004).190

8. B. Malorny, J. Hoorfar, C. Bunge and R. Helmuth, Appl Environ Microbiol 69, 290 (2003).191

9. B. Malorny, P.T. Tassios, P. Rdstrm, N. Cook, M. Wagner and J. Hoorfar, Int J Food192

Microbiol 83, 39 (2003).193

10. Anonymous,NV-DOC.D-20021022 (NordVal, Sborg, Denmark 2002).194

11. S. Qvist, Food Control 18, 113 (2007).195

12. K. Rahn, S.A. De Grandis, R.C. Clarke et al., Mol Cell Probes 6, 271 (1992).196

13. Agresti A,An introduction to categorical data analysis(John Wiley, New York, NJ 1996)197

14. Anonymous,ISO 16140 (International Organisation for Standardization, Geneva, Switzerland198

2003).199

15. Anonymous,NMKL procedure no. 20 (Nordic Method Commitee on Food Analysis (NMKL),200

Oslo, Norway 2007).201


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16. S. Chen, A. Yee, M. Griffiths et al., Int J Food Microbiol 35, 239 (1997).202

17. H.J. Beckers, J. vd Heide, U. Fenigsen-Narucka and R. Peters, J Appl Bacteriol 62, 97 (1987).203

18. W. Abu Al-Soud and P. Rdstrm, Appl Environ Microbiol 64, 3748 (1998).204

19. S. Perelle, F. Dilasser, B. Malorny, J. Grout, J. Hoorfar and P. Fach, Mol Cell Probes 18, 409205

(2004).206

20. P.F. Wolffs, K. Glencross, R. Thibaudeau and M.W. Griffiths, Appl Environ Microbiol 72,207

3896 (2006).208

21. I. Hein, G. Flekna, M. Krassnig and M. Wagner, J Microbiol Methods 66, 538 (2006).209

210


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Table 1 Comparison of the results in the comparative trial obtained by PCR and the reference culture methoda.211

Category Sample type PA NA FN TP FP Totalb

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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Total 68 169 6 4 2 249 96.0 97.3 98.8 0.88

aPA: 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 + FP213

bThe 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 was215

therefore 249.216

c-, calculation of SE was not possible because PA + FN = 0217


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61

62

63

64

65

15

FIGURE LEGEND218

Fig. 1. Overview of the validation study set-up.219220


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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

Löfström Et Al 2008 Pre-print FANM 1(1), 23-37

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