AEM Accepts, published online ahead of print on 15 August 2008 Appl. Environ. Microbiol. doi:10.1128/AEM.00405-08 Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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A real-time PCR method for detection of pathogenic

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Yersinia enterocolitica in Food

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S. Thisted Lambertz1*, C. Nilsson1, S. Hallanvuo2 and M. Lindblad1

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Research and Development Department, National Food Administration, Uppsala, Sweden1; Environmental and

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Food Research Laboratory (TavastLab), Municipal Joint Union for Public Health in Hämeenlinna Region,

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Hämeenlinna, Finland2

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*Corresponding author: Susanne Thisted Lambertz, National Food Administration

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Research and Development Department, PO Box 622, SE-751 26 Uppsala, Sweden

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Phone: +46 (0)18 17 55 62; Fax: +46 (0)18 17 14 94; E-mail: [email protected]

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Current methods for detection of pathogenic Y. enterocolitica in food are time-

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consuming and inefficient. Therefore, we have developed and in-house evaluated a

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TaqMan probe-based real-time PCR method for detection of this pathogen. The

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complete method comprised overnight enrichment, DNA extraction and real-time PCR

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amplification. Included in the method was also an internal amplification control. The

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selected primer-probe set was designed to use a 163-bp amplicon from the

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chromosomally located gene ail (attachment and invasion locus). The selectivity of the

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PCR was tested on a diverse range (n=152) of related and unrelated strains and no false-

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negative or false-positive PCR results were obtained. The sensitivity of the PCR

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amplification was 85 fg purified genomic DNA equivalent to 10 cells per PCR-reaction

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tube. Following enrichment of 10-g of food sample (milk, minced beef meat, cold-

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smoked sausage, fish and carrots) the sensitivity ranged from 0.5 to 55 cfu Y.

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enterocolitica. Good quality of precision, robustness and efficiency of the PCR

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amplification was also established. In addition, the method was tested on naturally contaminated food; in all 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as reference, the PCR products amplified from these samples were positively verified using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for detection of pathogenic Y.

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enterocolitica in food, as presented here, provide a superior alternative to the current

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available detection methods and makes it possible to identify the foods at risk of Y.

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enterocolitica contamination.

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Y. enterocolitica is causing yersiniosis in many countries in the world (28). Yersiniosis is an

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internal infection with the predominant symptoms being diarrhoea, fever, abdominal pain and

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vomiting. On a worldwide basis, the vast majority of reported cases occur sporadically, with

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Y. enterocolitica bio/serotype 4/O:3 being main responsible for the infections and with the

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source of infection unsolved. Y. enterocolitica is predominately considered as a food-borne

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agent, and as the bacterium has the ability to multiply in foods at low temperatures,

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approaching 0°C, as well as in vacuum- and modified atmosphere packages, it is of significant

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concern from a food safety and public health perspective.

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Y. enterocolitica is widely distributed in nature, however, among a large number of existing Y.

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enterocolitica bioserotypes only a few are pathogenic to humans (17, 28). The most

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commonly encountered bioserotypes isolated from yersiniosis patients worldwide are 4/O:3,

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2/O:9, 2/O:5:27 and 1B/O:8. It is well established that pigs are the main reservoir for Y.

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enterocolitica and pork is therefore likely to be the most important vehicle for the transmission to humans, directly or indirectly (17). However, the pathogen has seldom been isolated from food (8). The problem has been identified as methodological referring to the fact that no traditional culture method works satisfactory. Detection of Y. enterocolitica in food can be significantly improved by PCR, especially the second generation of PCR

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methodologies, i.e., real-time PCR, which has overcome several limitations of a conventional

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PCR, may be useful.

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In recent years, workers have developed PCR-based assays for detection of pathogenic Y.

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enterocolitica in food. One conventional PCR assay developed in our laboratory targets the

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chromosomally located virulence associated gene ail in a nested-PCR format (30). Other

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researchers have reported conventional PCR assays designed to target the ail gene as well as

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other genes using single or nested PCR formats (4, 18, 21). Virulence in Y. enterocolitica

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results from a complex interplay between a series of plasmid- and chromosomally located

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genes. However, PCR targets located on the virulence plasmid must be considered unsuitable

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as targets for detection because the plasmid is unstable and easily lost during laboratory

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treatment (5, 22).

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Real-time PCR assays, especially those using TaqMan-based probes, provide greater

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specificity and require less time and labour to complete than conventional PCR. Real-time

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PCR assays for detection of pathogenic Y. enterocolitica in food using a TaqMan probe have

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previously been developed (6, 16, 31). However, in some instances these methods are not

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applicable as a diagnostic tool as they were not rigorously evaluated or lack an internal

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amplification control.

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The European Standardization Committee (CEN) in collaboration with the International Standardization Organisation (ISO) is currently preparing a protocol describing minimal requirements of performance characteristics for molecular methods developed for detection of nucleic acid sequences (1). In addition, an EU-research project entitled FOOD-PCR has proposed a strategy and principles for standardization of PCR methods developed for

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detection of food-borne pathogens. An in-house evaluated PCR method should fulfil certain

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criteria such as high selectivity, sensitivity, precision, robustness etc. and an important

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prerequisite is that an internal amplification control is included in the method (1, 24). The aim

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of this study was to develop a method for detection of the pathogenic bioserotypes of Y.

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enterocolitica present in food by using real-time PCR with TaqMan probes applying

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performance criteria suggested by international standardization bodies for an in-house

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evaluated method.

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MATERIALS AND METHODS

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Bacterial strains and growth medium. The 152 bacterial strains used in this study are shown in Table 1. The

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strains were from human (n=81), pig (n=16), food (n=27), water (n=2) and unknown (n=26) sources. The

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pathogenic Y. enterocolitica strains were chosen to represent the most common bioserotypes associated with

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human or animal disease, and the non-pathogenic strains are the most common recovered from food, human, and

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environmental sources. The strains were recognised as pathogenic and non-pathogenic by virtue of their origin

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and/or biochemical classification. All strains were prepared as follows: pure colonies were grown on nutrient

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agar NA (CM3, Oxoid Hampshire, England) at 30°C overnight. Per sample, a loop of colonies were transferred

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to a tube containing 200 µl of MilliQ water (Millipore, Bedford, MA, USA) and 20 µl 0.8 M NaOH. The tubes

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were incubated for 10 min at 70 to 75°C, and subsequently 48 µl of equal volumes of 0.8 M HCl and 0.1 M Tris

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(pH 8.3) were added. The samples were mixed and centrifuged. Aliquots of 5 µl of each sample were used for

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the PCR amplification. The reference strain Y. enterocolitica bio/serotype 4/O:3 (SLV-408, CCUG 45643)

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originally isolated from frozen raw dog food (pig meat) was used for optimization and evaluation of the real-time

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C A PCR assay.

Terms. Selectivity is a measure of the degree of response from target and non-target microorganisms (1). It comprises the two terms (i) inclusivity, i.e. detection of the target organism from a wide range of strains, and (ii) exclusivity, i.e. lack of response from a relevant range of closely related non-target microorganisms. Sensitivity of a PCR assay is the lowest number of cells that can be detected in one single analysis, precision the closeness

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of agreement between test results and robustness refers to the ability of the PCR to withstand small procedural or

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environmental changes. Finally, accuracy refers to the closeness of agreement between a test results and the

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accepted reference value.

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DNA purification. When the PCR assay was tested for sensitivity, precision, robustness and detection

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probability, either enriched homogenates, broth cultures or cell suspensions were used and prior to the PCR

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amplification genomic DNA was extracted by using the DNeasy Blood and Tissue kit (Qiagen Gmbh, Hilden,

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Germany). The procedure was conducted according to the manufacturer’s protocol. One millilitre of the

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homogenates or broth cultures or 100-200 µl of the cell suspensions was used, and the final DNA was

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resuspended in 200 µl AE-buffer.

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Real-time PCR and cycling parameters. To find the primer concentrations that would produce the optimal

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amplification signal, nine different combinations between 50-900 nM of the forward and reverse primer were

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tested. The optimal PCR conditions were the following: 1x TaqMan®Universal PCR Master mix (contains

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AmpliTaq Gold DNA polymerase, dNTPs, Passive reference 1 and optimized buffer components; Applied

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Biosystems, Foster City, CA, USA), primers (real 9A and real 10A) to a final concentration of 900 nM, probe to

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a final concentration of 200 nM, approximately 100 copies of internal amplification control IAC-DNA

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(TaqMan® Exogenous Internal Positive Control; Applied Biosystems, Foster City, CA, USA), 1x IAC Mix

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(Applied Biosystems, Foster City, CA, USA) and, finally, 5-µl aliquots of the sample per reaction mixture.

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Sterile MilliQ water was used to adjust the volume of each reaction mixture to 25 µl. PCR cycling parameters

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were as follows: initial denaturation of the template DNA at 95°C for 10 min, followed by 45 cycles at 95°C for

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15 s and at 60°C for 1 min. In order to prevent carryover contamination, TaqMan® Universal PCR Master mix

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(Applied Biosystems, Foster City, CA, USA) with the inclusion of dUTP instead of dTTP was used in all tests

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on naturally contaminated foods presented in this paper; uracil-N-glycosylase (UNG) was then applied in the reaction to inactivate all undesired amplified PCR products (at an incubation step of 50°C for 2 min which was added to cycling parameters described above) prior to the amplification reaction. The analyses were performed

in 96-well plates (occasionally 8-strips) using the Applied Biosystems 7500 real time PCR system (Applied Biosystems, Foster City, CA, USA). A non-target control containing 5 µl of MilliQ water instead of DNA was

included in each run to detect any PCR fragment contamination. In addition, a non-patented variant of an internal

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amplification control, IAC, based on pUC19 (Fermentas, Germany) was tested. Thus, instead of the IPC system

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described above, 500 nM of each of the primers (IAC_fw and IAC_re; MWG Biotech, Germany), 200 nM of the

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belonging IAC probe (Applied Biosystems, Foster City, CA, USA) and 1 fg of pUC19 was used in each reaction

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mixture (information below).

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Internal amplification controls. Two internal amplification controls (named IPC and IAC, respectively) were

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tested in this study: (i) A commercially available TaqMan® Exogenous Internal Positive Control (Applied

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Biosystems, Foster City, CA, USA) was used. The reagent kit included primers, a Vic™probe, IPC target DNA

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and blocking solution. The IPC target DNA was diluted 10 times to achieve a copy number of approximately 100

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per PCR reaction. The PCR product length is not declared to the costumer. (ii) An open formula pUC 19 based

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internal amplification control IAC developed by Fricker et al. (9) was used. Approximately 50-100 copies of

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target DNA (pUC 19) were used per PCR reaction. The size of the IAC was 119 bp. The real-time PCR assay

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was optimized to work under conditions suitable for detection of the target organism and not the IACs; thus the

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IACs were let to follow that.

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Primer and probe design. One primer set and two alternative probes were used. Primers, R-real 9A:

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CCCAGTAATCCATAAAGGCTAACATAT (27-mer) and F-real 10A: ATGATA-

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ACTGGGGAGTAATAGGTTCG (26-mer); probes, ail probe: FAM-TGACCAAACTTATTACTGCCATA-

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MGB and Ye probe: FAM-TCTATGGCAGTAATAAGTTTGGTCACGGTGATCT-TAMRA. Two probes were

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used instead of one in order to offer the user multiple options with a non-patented so called open formula. The

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primer and probe sequences were designed manually and calculated using nearest-neighbour algorithm by using

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the Primer Express program (version 3.0) to achieve an annealing/extension temperature of 60°C. The specificity

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of the amplified sequence was tested by a BLAST search in GenBank database (http:/www.ncbi.nlm.nih.gov).

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Both probes were of TaqMan®-style; one was labelled at the 5´end with the reporter dye 6-carboxyfluorescein

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(FAM) and at the 3´end with a special chemical compound called minor groove binder (MGB). The other was also labelled at the 5´end with FAM but at the 3´end with the reporter dye tetramethyl-6-carboxyrhodamine (TAMRA). All primers and probes were purchased at www.appliedbiosystems.com.

Sequencing. PCR products amplified from seven of the pathogenic Y. enterocolitica strains representing five

different bio/serotypes, and PCR products amplified from nine of the naturally contaminated food samples were

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sequenced. The PCR products were purified using QIAquick® PCR Purification kit (Qiagen Gmbh, Hilden,

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Germany) according to the manufactures’ protocol before sequenced at Uppsala Genome Centre (Rudbeck

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Laboratory, SE-751 85 Uppsala, Sweden). Sequencing was performed in both directions.

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Sensitivity and detection probability. The sensitivity of the PCR amplification was determined with purified

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DNA. Purified genomic DNA of Y. enterocolitica (purified as described above) was serially diluted 10-fold in

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sterile MilliQ water and subjected to PCR amplification by using the reaction and cycling parameters described

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above. The DNA concentration was measured with a NanoDrop® ND-1000 spectrophotometer at a wavelength

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of 260 nm. In addition, to determine the detection probability, a broth culture of Y. enterocolitica (SLV-408)

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grown in brain heart infusion, BHI, (Oxoid, Hampshire, England) at 25°C overnight was 10-fold serially diluted

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in peptone water (Oxoid). Aliquots (0.1 ml) of the serial dilution were plated in duplicate onto NA plates and

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grown at 30°C overnight. The colonies were counted to determine the total number of viable Y. enterocolitica

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cfu. Genomic DNA was extracted (purified as described above) from 1 ml of each of the dilutions presumed to

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contain 100 to 106 cfu per ml. Per dilution, five aliquots (5 µl) and 100 copies of the IPC per aliquot were used

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for PCR amplification. This experiment was repeated six times, thus resulting in 30 PCR results for each cell

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concentration.

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Precision test. The precision of the PCR amplification was determined by analysing a 10-fold serial dilution of

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genomic DNA of Y. enterocolitica once on three consecutive days conducted by the same person. The samples

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were prepared as follows: colonies of Y. enterocolitica (SLV-408) were suspended in 1 ml of peptone water and

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genomic DNA released as described above. The DNA concentration was measured with a NanoDrop®;

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spectrophotometer at a wavelength of 260 nm. The extracted DNA was 10-fold serially diluted in sterile MilliQ

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water and aliquots (5 µl) of the appropriate dilutions to achieve 106, 105, 104, 103, 102, 101 and 100 genomic

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equivalents of Y. enterocolitica DNA were used for PCR amplification. All analyses were performed in the

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presence of 100 copies of the IPC. In parallel, the 10-fold serial dilution was used to produce a standard curve.

Robustness test. The robustness of the PCR amplification was determined by testing four different annealingextension temperatures, i.e. 58, 60, 62 and 64°C, and by using optimized and suboptimal concentrations, i.e. ±20 %, of the Universal PCR master mix. Each of the conditions was tested in eight replicates on 50 copies of genomic DNA of Y. enterocolitica (SLV-408) in the presence of 100 copies of the IAC. All other cycle conditions were kept constant. The samples were analysed in duplicates.

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Detection in a background of non-Y. enterocolitica DNA. A pure culture of Y. enterocolitica (SLV-408) was

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grown in BHI broth at 25°C overnight and genomic DNA released from 1 ml of the broth (as described above).

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The DNA concentration was determined spectrophotometrically before 10-fold serially diluted. In parallel a vial

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containing a freeze dried mixture of Enterobacter cloacae (8x103 cfu per ml), Campylobacter jejuni (30 cfu per

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ml), Escherichia coli O157 (40 cfu per ml), Listeria monocytogenes (60 cfu per ml) and Salmonella Dublin (15

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cfu per ml) were cultured in tryptone soya broth (TSB) at 25°C overnight. DNA was also extracted from this

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broth and the DNA concentration was determined spectrophotometrically. Equal volumes (2.5 µl) of the

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extracted background DNA and various concentrations of DNA from Y. enterocolitica ranging from 100 to 106

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genomic equivalents (per 2.5 µl) were mixed and amplified by PCR as described above. In parallel, the same

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concentrations 100 to 106 of DNA from Y. enterocolitica were subjected to PCR amplification alone, i.e. without

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presence of DNA from the background flora. All samples were analysed in duplicates.

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Detection in artificially inoculated food. A pure culture of Y. enterocolitica (SLV-408) was grown in BHI

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broth at 25°C overnight. The culture was 10-fold serially diluted in peptone water and the total number of viable

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cells was determined by viable plate count (as described above). One hundred microlitre of appropriate dilution

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was used to inoculate five different food items: milk (pasteurized, 1.5 % fat), raw minced beef, cold smoked

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sausage (heat treated at 65°C for 1 to 3 min), carrots (clean unpeeled) and raw fish. The food samples were

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purchased at local stores in Uppsala, Sweden, and transported and stored chilled until analysis which was started

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either the same day or the day after arrival. Five 10 gram portions of each food were aseptically transferred to

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sterile petri dishes and inoculated on the surface with Y. enterocolitica to provide cell numbers of 100, 101, 102,

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103 and 104 cfu per 10 gram of food. The inoculated foods were kept for 0.5 h at 25°C before being mixed with

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90 ml TSB supplemented with 0.6 % yeast extract (Y) and homogenised for 1 min (using Stomacher® Seward

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filter bags). The homogenates were enriched at 25°C for 18-20 h. DNA from 1 ml of the homogenates was released (as described above). An aliquot (5 µl) of each sample was used for PCR amplification. All samples were analysed in duplicates.

Detection in naturally contaminated food. One hundred five samples of food, comprising raw pork meat

(n=25), cold-smoked sausage (n=25), fish (n=25), carrots (n=25) and milk (n=5), were purchased from shops in

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Uppsala during June to August 2007. In addition, 20 raw milk samples were collected in June 2007 from milk

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cows on two dairy farms located near Uppsala. The food items were transported and stored chilled until analysis

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which was started either the same day or the day after arrival. Twenty-five gram portions of each food were

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homogenised in 225 ml TSB+Y and enriched at 25°C for 18-20 h. Each homogenate was gently mixed and

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coarse particles allowed to settle for 15-20 min before DNA from 1 ml of the homogenates was released (as

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described above). An aliquot (5 µl) of extracted DNA from each of the samples was used for PCR amplification.

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Statistical analysis. The Pearson product-moment correlation coefficient (r) was used to examine the relations

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between Ct values and log concentrations of concentrations of purified DNA and log numbers of Y.

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enterocolitica cells (without enrichment). A univariate general linear model (GLM) was used to analyze the

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relation between numbers of Y. enterocolitica cells inoculated to food and Ct values after enrichment, and

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possible effects of food type on the relation. The coefficient of variance was used as a measure of intra-

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individual day-to-day reproducibility in PCR amplifications of different concentrations of DNA.

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RESULTS

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Optimization and efficiency of real-time PCR. When nine different primer concentration

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combinations were tested 900 nM of each of the primers was found to produce the optimal

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amplification signal. A standard curve based on a 10-fold serial dilution between 101 and 106

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of genome equivalents of Y. enterocolitica DNA per PCR reaction showed a linear

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relationship between log input DNA and threshold cycles. The slope was -3.56 and the

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efficiency 91 %. The threshold line was set to a fluorescence value of 0.01 and was used

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Selectivity of detection. Real-time PCR amplification of genomic DNA using the primers

and probes, the reaction conditions and cycling parameters described here resulted in amplification of the expected 163 bp amplified fragment from all 102 isolates of the

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pathogenic Y. enterocolitica strains tested, i.e. 100 % inclusivity (Table 1). Seven of the

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amplicons were analysed on a 2 % agarose gel confirming a single fragment of the expected

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length compared to a known DNA size marker (Amersham Biosciences UK). The identity of

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PCR amplicons from these seven isolates were confirmed by sequencing (Table 2a). The

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sequence data showed that the bio/serotypes 4/O:3, O:9 and O:5,27 contained 100 % identical

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nucleotides over the fragments investigated. The amplified 1B/O:8 fragment differed from

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that in four nucleotides. The sequences were subjected to a homology search in BlastN which

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revealed no other identical sequences than those reported for the ail gene of Yersinia spp. No

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amplification was noticed with any of the 50 non-pathogenic Y. enterocolitica bioserotypes

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and other non-Yersinia species, thus the exclusivity was 100 %. The results reported here

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were equally valid for amplification by using the primers in combination with either the

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MGB-probe or the TAMRA-probe.

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Sensitivity and detection probability. The minimum level of detection of the ail target from

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purified genomic Y. enterocolitica DNA was 85 fg with a mean Ct value of 36.93 (standard

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deviation = 0.90). This level of detection was equivalent to 10 cells of Y. enterocolitica based

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on the assumption that single copies of the target are present in the genome (Table 3). The

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analysis of detection probability from a 10-fold serially diluted cell suspension of Y.

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enterocolitica revealed that there was amplification in 47 % (14 out of 30) of the samples with

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a cell count level of 10 Y. enterocolitica per ml and in 97 % (29 out of 30) of the samples with

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a cell count level of 100 Y. enterocolitica per ml. Highly significant correlations (Pearson`s r = -1.0, p