CEN ISO/TS 18867:2015

SLOVENSKI STANDARD
01-december-2015
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za
ugotavljanje prisotnosti patogenih mikroorganizmov v živilih - Ugotavljanje
prisotnosti patogenih Yersinia enterocolitica in Yersinia pseudotuberculosis
(ISO/DTS 18867:2015)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of
food-borne pathogens - Detection of pathogenic Yersinia enterocolitica and Yersinia
pseudotuberculosis (ISO/DTS 18867:2015)
Mikrobiologie der Lebensmittelkette - Polymerase-Kettenreaktion (PCR) zum Nachweis
von pathogenen Mikroorganismen in Lebensmitteln - Nachweis von pathogenen Yersinia
enterocolitica und Yersinia pseudotuberculosis (ISO/DTS 18867:2015)
Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR)
pour la détection de micro-organismes pathogènes dans les aliments - Détection des
Yersinia enterocolitica et Yersinia pseudotuberculosis pathogènes (ISO/DTS
18867:2015)
Ta slovenski standard je istoveten z: CEN ISO/TS 18867:2015
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN ISO/TS 18867
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
October 2015
TECHNISCHE SPEZIFIKATION
ICS
English Version
Microbiology of the food chain - Polymerase chain reaction
(PCR) for the detection of food-borne pathogens -
Detection of pathogenic Yersinia enterocolitica and
Yersinia pseudotuberculosis (ISO/TS 18867:2015)
Microbiologie de la chaîne alimentaire - Réaction de Mikrobiologie der Lebensmittelkette - Polymerase-
polymérisation en chaîne (PCR) pour la détection de Kettenreaktion (PCR) zum Nachweis von pathogenen
micro-organismes pathogènes dans les aliments - Mikroorganismen in Lebensmitteln - Nachweis von
Détection des Yersinia enterocolitica et Yersinia pathogenen Yersinia enterocolitica und Yersinia
pseudotuberculosis pathogènes (ISO/TS 18867:2015) pseudotuberculosis (ISO/TS 18867:2015)
This Technical Specification (CEN/TS) was approved by CEN on 29 May 2015 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 18867:2015 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (CEN ISO/TS 18867:2015) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent
rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO/TS 18867:2015 has been approved by CEN as CEN ISO/TS 18867:2015 without any
modification.
TECHNICAL ISO/TS
SPECIFICATION 18867
First edition
2015-09-15
Microbiology of the food chain —
Polymerase chain reaction (PCR)
for the detection of food-borne
pathogens — Detection of pathogenic
Yersinia enterocolitica and Yersinia
pseudotuberculosis
Microbiologie de la chaîne alimentaire — Réaction de polymérisation
en chaîne (PCR) pour la détection de micro-organismes pathogènes
dans les aliments — Détection des Yersinia enterocolitica et Yersinia
pseudotuberculosis pathogènes
Reference number
ISO/TS 18867:2015(E)
©
ISO 2015
ISO/TS 18867:2015(E)
© ISO 2015, Published in Switzerland
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of
the requester.
ISO copyright office
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CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles . 1
4.1 General . 1
4.2 Microbial enrichment . 2
4.3 Nucleic acid extraction . 2
4.4 Amplification and detection . 2
4.5 Isolation . 2
5 Reagents . 2
5.1 General . 2
5.2 Culture media . 2
5.2.1 General. 2
5.2.2 Diluent . 2
5.2.3 Enrichment media . 2
5.2.4 Selective solid medium . 4
5.2.5 Potassium hydroxide in saline solution, KOH . 5
5.3 Nucleic acid extraction . 5
5.4 Reagents for PCR . 5
5.5 Primers and probes. 5
6 Apparatus and equipment . 5
6.1 General . 5
6.2 Equipment for sample preparation prior to enrichment . 6
6.3 Equipment for microbial enrichment . . 6
6.4 Equipment for nucleic acid extraction . 6
6.5 Equipment for real-time PCR. 6
7 Sampling . 6
8 Procedure. 6
8.1 Sample preparation prior to enrichment . 6
8.1.1 General. 6
8.1.2 Preparation of the sample . 6
8.2 Microbial enrichment . 7
8.2.1 Pathogenic Y. enterocolitica . 7
8.2.2 Y. pseudotuberculosis . 7
8.2.3 Pathogenic Y. enterocolitica and Y. pseudotuberculosis . 7
8.3 Isolation of colonies, optional . 7
8.3.1 Pathogenic Y. enterocolitica . 7
8.3.2 Y. pseudotuberculosis . 7
8.3.3 Process controls . 8
8.4 Nucleic acid extraction . 8
8.5 PCR amplification . 8
8.5.1 General. 8
8.5.2 PCR controls . . 8
8.6 Confirmation of the PCR product . 8
8.6.1 General. 8
8.6.2 Interpretation of the PCR result . 8
9 Test report . 9
Annex A (normative) PCR detection and isolation of pathogenic Y. enterocolitica (see
ISO/TS 18867:2015(E)
Figure A.1) .10
Annex B (informative) Real-time PCR for detection of Y. enterocolitica .11
Annex C (informative) Detection and isolation of Y. pseudotuberculosis .21
Annex D (informative) Simultaneous detection of pathogenic Y. enterocolitica and
Y. pseudotuberculosis using multiplex real-time PCR .26
Bibliography .29
iv © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is the European Committee for Standardization (CEN)
Technical Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with Technical
Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the
Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
ISO/TS 18867:2015(E)
Introduction
Yersinia enterocolitica and Yersinia pseudotuberculosis are zoonotic bacterial pathogens causing food-
borne infection (yersiniosis) in humans worldwide. The main reservoir for pathogenic Y. enterocolitica
[3]
is domestic pigs and for Y. pseudotuberculosis a wide range of domestic and wild animals such as
[4]
rodents, deer, birds, and various farm animals serve as potential reservoirs. Some of the biotypes of Y.
enterocolitica are associated with human infection. In contrast, all Y. pseudotuberculosis are considered
[9]
potentially pathogenic to humans.
The chromosomally located gene ail (attachment invasion locus) is present in all bio(sero)types of
[8]
Y. enterocolitica associated with disease and a variant of it is also present in Y. pseudotuberculosis.
The ail gene is the target gene used for detection in this Technical Specification, and the developed
[7][8][13][14]
primer/probe sets target different sites of the ail gene for the two pathogens.
vi © ISO 2015 – All rights reserved

TECHNICAL SPECIFICATION ISO/TS 18867:2015(E)
Microbiology of the food chain — Polymerase chain
reaction (PCR) for the detection of food-borne pathogens
— Detection of pathogenic Yersinia enterocolitica and
Yersinia pseudotuberculosis
1 Scope
This Technical Specification specifies two horizontal methods for detection of the pathogenic
bioserotypes of Y. enterocolitica and one for detection of Y. pseudotuberculosis by using real-time PCR-
based methods. The described methods allow for the detection of the two pathogens in enrichments
and allow the isolation of colonies. Y. pestis, the causative agent of bubonic and pneumonic plague
harbours a variant of the ail gene as well and will be detected by the same primer/probe set as Y.
pseudotuberculosis. However, Y. pestis is normally not associated with food. This Technical Specification
is applicable to products for human consumption, animal feeding stuffs, and environmental samples.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887-1, Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination – Part 1: General rules for the preparation of the
initial suspension and decimal dilutions
ISO 10273, Microbiology of food and animal feeding stuffs — Horizontal method for the detection of
presumptive pathogenic Yersinia enterocolitica
ISO 20837, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — Requirements for sample preparation for qualitative detection
ISO 20838, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — Requirements for amplification and detection for qualitative methods
ISO 22119, Microbiology of food and animal feeding stuffs — Real-time polymerase chain reaction (PCR) for
the detection of food-borne pathogens — General requirements and definitions
ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — General requirements and definitions
3 Terms and definitions
For the purpose of this document, the following terms and definitions given in ISO 22174 and
ISO 22119 apply.
4 Principles
4.1 General
The method comprises the following consecutive steps:
a) Microbial enrichment (4.2);
ISO/TS 18867:2015(E)
b) Nucleic acid extraction (4.3);
c) Amplification and detection (4.4);
d) Isolation (4.5).
4.2 Microbial enrichment
The number of pathogenic Y. enterocolitica and Y. pseudotuberculosis bacterial cells is increased by
growth in a non-selective or semi-selective liquid nutrient medium.
4.3 Nucleic acid extraction
Bacteria cells are separated from the nutrient broth, lysed, and the nucleic acid extracted for use in the
PCR reaction.
4.4 Amplification and detection
The extracted nucleic acid is amplified using a probe-based real-time PCR. Detection of the target sequence
is achieved by monitoring a clear increase in the fluorescence signal above the cycle threshold, Ct.
NOTE Probe-based real-time PCR combines amplification, detection, and confirmation of the target DNA.
4.5 Isolation
After a PCR-positive result is obtained, the target organism can be isolated by using culture methods as
described in this Technical Specification.
5 Reagents
5.1 General
For the stages in 4.1 b)-c), molecular grade reagents and consumables suitable for molecular biology
shall be used as given in ISO 20837 and ISO 20838.
Requirements are specified in ISO 20838.
The following media and reagents should be used.
5.2 Culture media
5.2.1 General
See ISO 7218 and ISO 11133 for the preparation, production, and performance testing of culture media.
5.2.2 Diluent
See ISO 6887-1 and the relevant part of ISO 6887 dealing with the product to be examined.
5.2.3 Enrichment media
5.2.3.1 Tryptone-soya broth supplemented with yeast, TSBY
5.2.3.1.1 Composition
Pancreatic digest of casein 17,0 g
2 © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
Papaic digest of soyabean meal 3,0 g
Sodium chloride, (NaCl) 5,0 g
Dibasic potassium phosphate, (K HPO ) 2,0 g
2 4
Glucose 2,5 g
Yeast extract 6,0 g
Water 1 000 ml
5.2.3.1.2 Preparation
Dissolve the above ingredients in 1 000 ml distilled water. Adjust the pH, if necessary, so that after
sterilization it is pH 7,3 ± 0,2. Dispense the medium into tubes or flasks of suitable capacity to obtain
portions appropriate for the test samples. Sterilize for 15 min at 121 °C ± 1 °C.
Store the medium in the dark at room temperature and not longer than 4 weeks.
Alternatively, use dehydrated Tryptone soya broth (TSB) 30 g/l supplemented with 0,6 % yeast extract,
pH 7,3 ± 0,2.
[15]
5.2.3.2 Peptone-sorbitol-bile-salt broth, PSB
5.2.3.2.1 Composition
Peptone 5,0 g
Sorbitol 10,0 g
Sodium chloride, (NaCl) 5,0 g
Disodium hydrogen phosphate (Na HPO ) 8,23 g
2 4
Sodium dihydrogen phosphate monohydrate (NaH PO .H O) 1,2 g
2 4 2
Bile salts 1,5 g
Water 1 000 ml
5.2.3.2.2 Preparation
Dissolve the components or the dehydrated complete medium in the water, if necessary by heating.
Adjust the pH, if necessary, so that after sterilization it is pH 7,6 ± 0,2.
Dispense the medium into tubes or flasks of suitable capacity to obtain portions appropriate for the
test samples. Sterilize for 15 min at 121 °C ± 1 °C.
[5]
5.2.3.3 Cold enrichment broth, PMB
5.2.3.3.1 Composition
Disodium hydrogen phosphate (Na HPO ) 7,6 g
2 4
Potassium dihydrogen phosphate (KH PO ) 1,0 g
2 4
Sodium chloride (NaCl) 8,5 g
ISO/TS 18867:2015(E)
Mannitol 10,0 g
Bile salts N°. 3 1,5 g
Water 1 000 ml
5.2.3.3.2 Preparation
Dissolve the components or the dehydrated complete medium in the water, if necessary by heating.
Adjust the pH, if necessary, so that after sterilization it is pH 7,6 ± 0,2.
Dispense the medium into tubes or flasks of suitable capacity to obtain portions appropriate for the
test samples. Sterilize for 15 min at 121 °C ± 1 °C.
5.2.4 Selective solid medium
[10]
5.2.4.1 Cefsulodin Irgasan Novobiocin agar, CIN
5.2.4.1.1 Basic medium, composition
Enzymatic digest of gelatin 17,0 g
Enzymatic digest of casein and animal tissues 3,0 g
Yeast extract 2,0 g
Mannitol 20,0 g
Sodium pyruvate 2,0 g
Sodium chloride, (NaCl) 1,0 g
Magnesium sulfate, (MgSO .7 H O) 0,01 g
4 2
Sodium desoxycholate 0,5 g
Neutral red 0,03 g
Crystal violet 0,001 g
Agar 12,5 g
Water 1 000 ml
5.2.4.1.2 Preparation
Dissolve the components or dehydrated basic medium in the water by boiling. Adjust the pH, if necessary,
so that after sterilization it is pH 7,4 ± 0,2 at 25 °C. Dispense the medium into flasks of suitable capacity.
Sterilize for 15 min at 121 °C ± 1 °C.
5.2.4.2 Supplements
5.2.4.2.1 Cefsulodin solution (15 mg/ml)
Dissolve 1,5 g Cefsulodin in 100 ml water. Sterilize by filtration.
™
5.2.4.2.2 Irgasan [5-chloro-2-(2,4-dichlorophenoxy)phenol], ethanolic solution (4 mg/ml).
Dissolve Irgasan in ethanol, store the solution at about −20 °C for not more than 4 weeks.
4 © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
5.2.4.2.3 Novobiocin solution (2,5 mg/ml)
Dissolve novobiocin in water. Sterilize by filtration.
5.2.4.3 Composition of the complete medium
Basic medium (5.2.4.1.1) 997 ml
Cefsulodin solution (5.2.4.2.1) 1 ml
Irgasan solution (5.2.4.2.2) 1 ml
Novobiocin solution (5.2.4.2.3) 1 ml
5.2.4.4 Preparation
Add each antibiotic solution aseptically to the basic medium, cooled to about 45 °C, and mix. Pour
approximately 15 ml of the complete medium into sterile petri dishes.
5.2.5 Potassium hydroxide in saline solution, KOH
5.2.5.1 Composition
Potassium hydroxide (KOH) 0,25 g/0,50 g
Saline solution 100 ml
NOTE It is recommended to use freshly prepared 0,5 % KOH for pathogenic Y. enterocolitica and 0,25 % for
Y. pseudotuberculosis.
5.2.5.2 Preparation
Dissolve the potassium hydroxide in the saline solution. Dispense the solution into flasks of a suitable
capacity. Sterilize for 15 min at 121 °C ± 1 °C. Prepare the solution the day before use.
5.3 Nucleic acid extraction
Nucleic acid extraction procedure and reagents appropriate for Gram-negative bacteria shall be used.
NOTE Commercial kits can also be used.
5.4 Reagents for PCR
See ISO 22119 and ISO 20838.
5.5 Primers and probes
The primers and probes for detection of pathogenic Y. enterocolitica and Y. pseudotuberculosis are listed
in Annex B and Annex C.
6 Apparatus and equipment
6.1 General
Microbiology equipment (see ISO 7218, ISO 20837, and ISO 22119), in particular, the following
ISO/TS 18867:2015(E)
6.2 Equipment for sample preparation prior to enrichment
Peristaltic blender and sterile bags with filter.
NOTE Filter of small pore size suitable for PCR is recommended.
6.3 Equipment for microbial enrichment
Incubators, capable of operating at 25 °C ± 1 °C and 30 °C ± 1 °C.
6.4 Equipment for nucleic acid extraction
6.4.1 Micro-centrifuge tubes, with capacities of 1,5 ml and 2,0 ml.
6.4.2 Centrifuge, for reaction tubes with a capacity of 1,5 ml and 2,0 ml and capable of achieving an
acceleration up to approximately 14 000 × g.
6.4.3 Thermoblock, with heating capacity of up to 100 °C.
6.4.4 Graduated pipettes and pipette filter tips, for volumes between 1 µl to 1 000 µl.
6.4.5 Mixer.
6.5 Equipment for real-time PCR
6.5.1 Real-time PCR thermal cycler.
6.5.2 96-well plates and/or 8-well strips.
7 Sampling
Sampling is not part of the method specified in this Technical Specification. If there is no specific
International Standard dealing with sampling of the product concerned, it is recommended that the
parties concerned come to an agreement on this subject.
8 Procedure
See diagram in Annex A.
8.1 Sample preparation prior to enrichment
8.1.1 General
It is recommended to analyse at least 25 g or 25 ml of sample. However, if sample amount is limited
other sample sizes can be used.
8.1.2 Preparation of the sample
Prepare and homogenize the sample according to ISO 6887-1 and the relevant part of ISO 6887 dealing
with the specific product type intended for analysis (see ISO 6887-1 to ISO 6887-5).
A test portion of the sample is added to the enrichment medium to obtain a ratio of the test portion to
medium of 1/10.
6 © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
8.2 Microbial enrichment
Depending on the target, most suitable enrichment medium should be used.
8.2.1 Pathogenic Y. enterocolitica
PSB enrichment medium should be used prior to detection by PCR.
PSB enrichment is performed at 25 °C ± 1 °C for 24 h ± 3 h.
If isolation of colonies is required, enrichment time should be increased up to 48 h. It is recommended
to refer to ISO 10273.
If thermal lysis is used to extract DNA (method Annex B), prolonged enrichment for 48 h in PSB
may be needed.
8.2.2 Y. pseudotuberculosis
TSBY enrichment medium should be used prior to detection by PCR.
TSBY enrichment is performed at 25 °C ± 1 °C for 24 h ± 3 h.
NOTE The procedure does not allow isolation of the bacterium. Isolation involves re-enrichment of the
sample by selective culture as described in 8.3.2 and C.2.
If there is a limited amount of sample or swab sample that cannot be divided for PCR screening (TSBY)
and isolation of colonies, PMB should be used instead of TSBY, see C.2, 5.2.3.3, and 8.3.2.
8.2.3 Pathogenic Y. enterocolitica and Y. pseudotuberculosis
For the simultaneous detection of Y. pseudotuberculosis and pathogenic Y. enterocolitica by PCR, TSBY
medium may be used.
The risk of false-negative results for Y. enterocolitica may increase when TSBY is used.
8.3 Isolation of colonies, optional
8.3.1 Pathogenic Y. enterocolitica
After enrichment for 24 h ± 3 h, transfer 0,5 ml of the PSB enrichment into 4,5 ml 0,5 % potassium
hydroxide solution, KOH (5.2.5) and mix gently for 25 s ± 5 s. Inoculate 1 µl, 10 µl, and 100 µl of the
mixture after streaking on the surface of three CIN-agar plates. Continue incubation of the enrichment
for further 24 h. Incubate CIN agar plates at 30 °C ± 1 °C for 24 h ± 3 h. Plates are examined (preferably
by stereo microscope) and typical colonies with bull’s eye appearance are picked for confirmation.
Second plating on CIN agar is made after 48 h ± 4 h enrichment, performed in the same way as for the
plating after 24 h.
Presumptive pathogenic Y. enterocolitica colonies should be confirmed according to ISO 10273.
NOTE Only for samples with high expected Y. enterocolitica contamination levels, enrichment in PSB can
be used to isolate colonies as described in this Technical Specification. For samples with low suspected Y.
enterocolitica contamination levels, it is recommended to follow ISO 10273 for isolation of colonies.
8.3.2 Y. pseudotuberculosis
Test sample is inoculated (dilution 1/10) into the cold enrichment medium PMB (5.2.3.3), homogenized
and incubated at 4 °C ± 1 °C for 7 days and 14 days. If PCR detection of Y. pseudotuberculosis has to be
performed after enrichment in PMB (see 8.2.2), nucleid acid extraction and further PCR amplification
should be carried out after 72 h ± 8 h of incubation (see 8.4 and 8.5).
ISO/TS 18867:2015(E)
Transfer 0,5 ml of the incubated enrichment (PMB) into 4,5 ml 0,25 % potassium hydroxide solution,
KOH, (5.2.5) and mix gently for 25 s ± 5 s. Transfer the mixture by using a 10 µl loop onto the surface of
a CIN-agar plate(performed in duplicate). Incubate CIN-agar plates at 30 °C ± 1 °C for a total of 48 h ± 4 h
but examine the plates for typical growth after 24 h. Plates are examined by stereo microscope and
typical colonies are picked for confirmation. Typical colonies are small (usually <0,5 mm in diameter
after 24 h incubation) and irregular-edged. Colonies usually have deep-red diffuse centre with no clear
borderline. The transparent or translucent zone surrounding the centre has ground-glass appearance
and may be thin and hardly visible after 24 h. For confirmation, biochemical or PCR tests can be used.
8.3.3 Process controls
Positive and negative process controls shall be used according to ISO 22174.
8.4 Nucleic acid extraction
Nucleic acid is extracted from a portion, usually 1 ml, of the enriched culture. Any nucleic acid extraction
procedure appropriate for Gram-negative bacteria tested suitable for this purpose can be used, for
[2]
example the CTAB extraction method. Also commercial kits can be used.
NOTE It is known that for certain matrices a nucleic acid extraction method producing high yield and high
purity DNA is needed (see Annexes B, C, and D for the tested matrices).
8.5 PCR amplification
8.5.1 General
Different protocols for probe-based real-time PCR amplification can be used.
Examples of real-time PCR assays for detection of pathogenic Y. enterocolitica are given in Annex B.
Example of a real-time PCR assay for Y. pseudotuberculosis is given in Annex C.
Example of a real-time PCR assay for simultaneous detection of pathogenic Y. enterocolitica and
Y. pseudotuberculosis is given in Annex D.
8.5.2 PCR controls
PCR controls shall be in accordance with ISO 22174.
8.6 Confirmation of the PCR product
8.6.1 General
According to ISO 20838 a probe-based PCR-positive result does not require additional confirmation of
the PCR product.
8.6.2 Interpretation of the PCR result
The result obtained, including the controls specified in ISO 22174, should be unambiguous otherwise
the PCR shall be repeated.
The PCR result will be either
a) positive if a specific PCR product has been detected and all the controls give expected results, or
b) negative within the limits of detection, if a specific PCR product has not been detected, and all
controls give expected results.
8 © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
9 Test report
The test report shall conform to the requirements of ISO 22174.
Additionally, it shall specify which method within this Technical Specification has been used, in particular
when the simultaneous detection of pathogenic Y. enterolitica and Y. pseudotuberculosis is performed.
ISO/TS 18867:2015(E)
Annex A
(normative)
PCR detection and isolation of pathogenic Y. enterocolitica (see
Figure A.1)
Figure A.1 — Flow diagram for PCR detection and isolation of pathogenic Y. enterocolitica
10 © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
Annex B
(informative)
Real-time PCR for detection of Y. enterocolitica
B.1 Real-time PCR for detection of Y. enterocolitica — Method 1
B.1.1 General
This is a method for the detection of pathogenic Yersinia enterocolitica by amplification of a sequence
specific for the ail locus of Y. enterocolitica. This gene is part of the pathogenicity mechanism of Y.
enterocolitica.
The detection system was developed as duplex real-time PCR in combination with a heterologous internal
amplification control based on the plasmid pUC 19. By real-time PCR, a fragment spanning from the lac
operon from M13mp18 to sequences of pBR322 is amplified. This sequence does not occur naturally.
B.1.2 Performance characteristics
B.1.2.1 General
The method is applicable for the detection of Y. enterocolitica strains representing the bioserotypes
associated with pathogenicity in humans.
[7]
The method has been published.
B.1.2.2 Selectivity
Selectivity was performed using the primers ye-ail-F2 and ye-ail-R2, in combination with the hydrolysis
probe ye-ail-tmp in combination with the internal amplification control using the primer-probe system
pUC 18-F, pUC 18-R, and Tm-pUC 18.
B.1.2.2.1 Inclusivity test
Inclusivity of the PCR assay was tested on 50 strains, 100 % inclusivity was obtained (see Table B.1).
The strains tested were isolated from samples of human, animal, and food origin.
Table B.1 — Inclusivity using 50 target strains
Species and bio/serotype Number of strains resulting in
(Number of strains) amplification of the target fragment
Y. enterocolitica 4/O:3 (33) 33
Y. enterocolitica 2/O:9 (3) 3
Y. enterocolitica 2/O:9 (11) 11
Y. enterocolitica 1B/O:8 (1) 1
Y. enterocolitica 2/O:5,27 (2) 2
Total (50) 50
ISO/TS 18867:2015(E)
B.1.2.2.2 Exclusivity test
Exclusivity of the PCR assay was tested on 51 non-target strains, 100 % exclusivity was obtained (see
Table B.2) The strains tested were isolated from samples of human, animal, and food origin.
Table B.2 — Exclusivity using 51 non-target strains
Number of strains resulting in
Species and bio/serotype
amplification of the target frag-
(Number of strains)
ment
Y. enterocolitica 1A/O:5 (2) 0
Y. enterocolitica 1A/O:6, 30 (1) 0
Y. enterocolitica 1A/O:10 (1) 0
Y. frederiksenii (2) 0
Y. kristensenii (2) 0
Y. intermedia (2) 0
Y. aldovae (1) 0
Y. mollaretii (1) 0
Y. pseudotuberculosis (3) 0
Diverse food-related bacterial species (34) 0
Saccharomyces cerevisiae (1) 0
Aspergillus niger (1) 0
Total (51) 0
B.1.2.2.3 Molecular selectivity
According to a BLAST search (16.5.2011) the primers and probe have been validated in silico.
B.1.2.3 Sensitivity
B.1.2.3.1 Reaction sensitivity
The limit of detection is 5 genome equivalents [25 femtograms (fg)] per single reaction. All analyses
were performed in the presence of approximately 25 copies of the internal amplification control (IAC).
B.1.2.3.2 Method sensitivity
[17]
The detection level LOD of the PCR method was 10,7 cfu per 25 g of food. The limit of detection
of the method was assessed measuring four different food matrices, i.e. Mortadella sausage, cabbage,
pasteurized milk, and uncooked fish, inoculated with 5 cfu and 50 cfu in 25 g with six replicates for
each contamination level. After 48 h of enrichment in PSB, the limit of detection in Mortadella sausage,
uncooked fish, and pasteurized milk was 5 cfu in 25 g and in cabbage it was 50 cfu in 25 g.
B.1.2.3.3 Performance parameters relative accuracy, relative sensitivity, relative specificity
Relative accuracy AC 89,5 %
Relative sensitivity SE 100 %
Relative specificity SP 76,7 %
12 © ISO 2015 – All rights reserved

ISO/TS 18867:2015(E)
The performance parameters were assessed by comparison with the culture method according to
ISO 10273:2003, Clause 5.
NOTE These data were obtained by the analysis of artificially contaminated samples.
B.1.2.4 Robustness
The robustness of the PCR was tested by applying the following modifications in the reaction setup.
— Increasing and decreasing the primer and probe concentrations by ±20 % in the presence of 25
copies of the internal amplification control.
— Use of two different formulations of the Taq-polymerase mastermixes from two suppliers.
The modifications did not influence the reaction performance.
B.1.2.5 Instruments
1)
Evaluation was carried out using the Roche LightCycler® 480 and the ABI 7900HT real-time PCR
1)
instruments .
Different real-time PCR instruments did not have a significant influence in the method’s performance.
B.1.3 Procedure
B.1.3.1 Principle
A specific DNA fragment of the ail locus of Yersinia enterocolitica is amplified and detected by real-
time PCR. The real-time PCR system is based on a specific hydrolysis probe which is labelled with
Carboxyfluorescein (FAM) as reporter molecule and Dabcyl as non-fluorescent quencher molecule.
B.1.3.2 Reagents
B.1.3.2.1 General
For quality of reagents used, see ISO 22174.
B.1.3.2.2 Reagents for PCR
See ISO 22119 and ISO 20838.
B.1.3.2.3 Oligonucleotides
B.1.3.2.3.1 Oligonucleotides, pathogenic Y. enterocolitica
Table B.3 — Sequences of the oligonucleotides
Primer and Size of the PCR
DNA sequence of the oligonucleotide (5’ – 3’)
probe product
ye-ail-F2 5’-GGT TAT GCA CAA AGC CAT GTA AA -3’
93 bp
ye-ail-R2 5’-AAA CGA ACC TAT TAC TCC CCA GTT-3’
a
FAM: 6 Carboxyfluorescein, DB: Dabcyl.
1) The Roche LightCycler® 480 and the ABI 7900HT real-time PCR instruments, are examples of suitable products
available commercially from Roche Diagnostics and Life Technologies respectively. This information is given for the
convenience of the user of this Technical Specification and does not constitute an endorsement of these products.
Equivalent products can be used if they can be shown to lead to equivalent results.
SIST-TS CE
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