SIST-TS CEN ISO/TS 18867:2015

SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 18867:2015
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
SIST-TS CEN ISO/TS 18867:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN ISO/TS 18867:2015

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SIST-TS CEN ISO/TS 18867:2015


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.

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CEN ISO/TS 18867:2015 (E)
Contents Page
European foreword . 3

2

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SIST-TS CEN ISO/TS 18867:2015
CEN ISO/TS 18867:2015 (E)
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.
3

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SIST-TS CEN ISO/TS 18867:2015
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

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ISO/TS 18867:2015(E)

COPYRIGHT PROTECTED DOCUMENT
© 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
Ch. de Blandonnet 8 • CP 401
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

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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
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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
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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).
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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.
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SIST-TS CEN ISO/TS 18867:2015
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);
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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
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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
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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.
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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 1
...