SIST EN ISO 6579-4:2025
(Main)Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 4: Identification of monophasic Salmonella Typhimurium (1,4,[5],12:i:-) by polymerase chain reaction (PCR) (ISO 6579-4:2025)
Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 4: Identification of monophasic Salmonella Typhimurium (1,4,[5],12:i:-) by polymerase chain reaction (PCR) (ISO 6579-4:2025)
This document specifies a horizontal in vitro method for the molecular identification and differentiation of the monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium (1,4,[5],12:i:-) lacking the second H phase H:1,2, starting from isolates. The method detects specific DNA sequences of an intergenic region of the first H phase flagellin cluster for identification of Salmonella enterica subsp. enterica serovar Typhimurium (further called Salmonella Typhimurium) and specific DNA sequences of genes associated with second H phase flagellar antigen expression.
The method is applicable for:
— differentiation of the isolate under analysis between monophasic Salmonella Typhimurium and the monophasic variant of another Salmonella non-Typhimurium serovar that has the same antigenic formula;
— identification of the isolate under analysis being either monophasic Salmonella Typhimurium or (biphasic) Salmonella Typhimurium.
This document is applicable for the analysis of a pure culture belonging to the genus Salmonella, isolated from:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.
This document can also be applied in other domains for identification of monophasic Salmonella Typhimurium (e.g. environmental, human health, animal health).
NOTE This method has been validated in a method evaluation study and in an interlaboratory study with a large set of different strains (target and non-target strains), isolated from different sources (food products, animals, animal feed, primary production samples and humans). For detailed information on the validation, see Annex E.
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis von Salmonella spp. - Teil 4: Identifizierung von monophasischen Salmonella Typhimurium (1,4,[5],12:i:-) durch Polymerase‑Kettenreaktion (PCR) (ISO 6579-4:2025)
Dieses Dokument legt ein horizontales, mit Isolaten beginnendes, In-vitro-Verfahren für die molekulare Identifizierung und Differenzierung der monophasischen Variante von Salmonella enterica subsp. enterica Serovar Typhimurium (1,4[5],12:i: ) fest, bei der die zweite H-Phase H:1,2 fehlt. Dieses Verfahren weist spezifische DNA-Sequenzen einer intergenischen Region der ersten H-Phase des Flagellin-Clusters nach, um Salmonella enterica subsp. enterica Serovar Typhimurium (im folgenden Salmonella Typhimurium) sowie spezifische DNA-Sequenzen von Genen, die mit der Expression des Flagellen-Antigens der zweiten H-Phase verbunden sind, zu identifizieren.
Das Verfahren ist anwendbar zur:
Differenzierung des untersuchten Isolats zwischen monophasischem Salmonella Typhimurium und der monophasischen Variante eines anderen Salmonella Nicht-Typhimurium-Serovars, der die gleiche Antigenformel hat;
Identifizierung des untersuchten Isolats, das entweder ein monophasischer Salmonella Typhimurium oder ein (biphasischer) Salmonella Typhimurium ist.
Dieses Dokument ist anwendbar für die Untersuchung einer zur Gattung Salmonella gehörenden Reinkultur, welche isoliert wurde aus:
Produkten, die für den menschlichen Verzehr vorgesehen sind;
Produkten, die als Futtermittel vorgesehen sind;
Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln;
Proben aus dem Bereich der Primärproduktion.
Dieses Dokument kann auch in anderen Bereichen (z. B. Umwelt, Gesundheit von Menschen, Gesundheit von Tieren) zur Identifizierung von monophasischem Salmonella Typhimurium angewandt werden.
ANMERKUNG Dieses Verfahren wurde in einer Studie zur Verfahrensbewertung sowie in einem Ringversuch mit einem umfangreichen Satz unterschiedlicher Stämme (Ziel- und Nicht-Ziel-Stämme) validiert, die aus verschiedenen Quellen (Lebensmittelerzeugnisse, Tiere, Futtermittel, Primärproduktionsproben und Menschen) isoliert wurden. Detaillierte Informationen zur Validierung siehe Anhang E.
WARNUNG — Zum Schutz der Gesundheit des Laborpersonals ist es unerlässlich, dass Untersuchungen zum Nachweis, zur Zählung und zur (Sero)Typisierung von Salmonella nur in ordnungsgemäß ausgestatteten Laboren unter Aufsicht eines qualifizierten Mikrobiologen vorgenommen werden und dass das gesamte bebrütete Material mit größter Sorgfalt entsorgt wird. Personen, die dieses Dokument anwenden, sollten mit üblicher Laborpraxis vertraut sein. Dieses Dokument erhebt nicht den Anspruch, dass alle mit seiner Anwendung verbundenen Sicherheitsaspekte angesprochen werden. Es obliegt der Verantwortung des Anwenders, angemessene Sicherheits- und Gesundheitsschutzmaßnahmen zu treffen.
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche, le dénombrement et le sérotypage des Salmonella - Partie 4: Identification du variant monophasique de Salmonella Typhimurium (1,4,[5],12:i:-) par réaction de polymérisation en chaîne (PCR) (ISO 6579-4:2025)
Le présent document spécifie une méthode horizontale in vitro d’identification moléculaire et de différenciation du variant monophasique de Salmonella enterica sous-espèce enterica sérotype Typhimurium (1,4,[5],12:i:-) ne présentant pas l’antigène H de seconde phase H:1,2, à partir d’isolats. La méthode détecte des séquences d’ADN spécifiques d’une région intergénique du groupe des flagellines de première phase H pour l’identification de Salmonella enterica sous-espèce enterica sérotype Typhimurium (encore appelé Salmonella Typhimurium) et des séquences d’ADN spécifiques de gènes associés à l’expression d’antigène flagellaire H de seconde phase.
La méthode est applicable pour:
— la différenciation de l’isolat analysé entre le variant monophasique de Salmonella Typhimurium et le variant monophasique d’un autre sérotype de Salmonella non Typhimurium ayant la même formule antigénique;
— l’identification de l’isolat analysé comme étant soit le variant monophasique de Salmonella Typhimurium, soit Salmonella Typhimurium (biphasique).
Le présent document est applicable à l’analyse d’une culture pure appartenant au genre Salmonella, isolée à partir:
— de produits destinés à la consommation humaine;
— de produits destinés à l’alimentation animale;
— d’échantillons environnementaux dans le domaine de la production et de la manutention de denrées alimentaires;
— d’échantillons au stade de la production primaire.
Le présent document peut aussi être appliqué dans d’autres domaines pour l’identification du variant monophasique de Salmonella Typhimurium (par exemple, domaine environnemental, santé humaine, santé animale).
NOTE La présente méthode a été validée dans la cadre d’une étude d’évaluation de méthode et d’une étude interlaboratoires à l’aide d’un large panel de souches différentes (souches cibles et non cibles), isolées à partir de différentes sources (produits alimentaires, animaux, aliments pour animaux, échantillons de production primaire et échantillons humains). Pour de plus amples informations sur la validation, voir Annexe E.
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti, štetja in serotipizacije Salmonella - 4. del: Identifikacija monofazne Salmonella Typhimurium (1,4,[5],12:i:-) s polimerazno verižno reakcijo (PCR) (ISO 6579-4:2025)
Ta dokument določa horizontalno metodo in vitro za molekularno identifikacijo in diferenciacijo monofazne variante Salmonella enterica podvr. enterica serovar Typhimurium (1,4,[5],12:i:-), ki nima druge faze H-antigena (H:1,2), iz izolatov. S to metodo se odkrivajo določena zaporedja DNK medgenske regije flagelinov prve faze H-antigena za identifikacijo Salmonella enterica podvr. enterica serovar Typhimurium (v nadaljevanju »Salmonella Typhimurium«) in določena zaporedja DNK genov, povezanih z ekspresijo flagelarnega antigena druge faze.
Metoda se uporablja za:
– diferenciacijo analiziranega izolata med monofazno Salmonella Typhimurium in monofazno varianto Salmonella drugega serovara z isto antigensko formulo;
– identifikacijo analiziranega izolata, tj. monofazne Salmonella Typhimurium oziroma (dvofazne) Salmonella Typhimurium.
Ta dokument se uporablja za analizo čiste kulture iz rodu Salmonella, izolirane iz:
– proizvodov, namenjenih za prehrano ljudi;
– proizvodov, namenjenih za živalsko krmo;
– okoljskih vzorcev v območju proizvodnje in ravnanja z živili in krmo;
– vzorcev iz faze primarne proizvodnje.
Ta dokument je mogoče uporabiti za identifikacijo monofazne Salmonella Typhimurium tudi na drugih področjih (npr. okolje, zdravje ljudi, zdravje živali).
OPOMBA: Ta metoda je bila validirana v študiji ocenjevanja metode in v medlaboratorijski študiji z velikim naborom različnih (ciljnih in neciljnih) sevov, izoliranih iz različnih virov (živilski proizvodi, živali, živalska krma, vzorci iz primarne proizvodnje in ljudje). Za podrobnejše informacije o validaciji glej dodatek D.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-april-2025
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti, štetja in serotipizacije Salmonella - 4. del: Identifikacija monofazne
Salmonella Typhimurium (1,4,[5],12:i:-) s polimerazno verižno reakcijo (PCR) (ISO
6579-4:2025)
Microbiology of the food chain - Horizontal method for the detection, enumeration and
serotyping of Salmonella - Part 4: Identification of monophasic Salmonella Typhimurium
(1,4,[5],12:i:-) by polymerase chain reaction (PCR) (ISO 6579-4:2025)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zum Nachweis von
Salmonella spp. - Teil 4: Identifizierung von monophasischen Salmonella Typhimurium
(1,4,[5],12:i:-) durch Polymerase‑Kettenreaktion (PCR) (ISO 6579-4:2025)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche, le
dénombrement et le sérotypage des Salmonella - Partie 4: Identification du variant
monophasique de Salmonella Typhimurium (1,4,[5],12:i:-) par réaction de polymérisation
en chaîne (PCR) (ISO 6579-4:2025)
Ta slovenski standard je istoveten z: EN ISO 6579-4:2025
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 6579-4
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2025
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Horizontal method for the
detection, enumeration and serotyping of Salmonella - Part
4: Identification of monophasic Salmonella Typhimurium
(1,4,[5],12:i:-) by polymerase chain reaction (PCR) (ISO
6579-4:2025)
Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche, le dénombrement et le Verfahren zum Nachweis, zur Zählung und zur
sérotypage des Salmonella - Partie 4: Identification du Serotypisierung von Salmonellen - Teil 4:
variant monophasique de Salmonella Typhimurium Identifizierung von monophasischen Salmonella
(1,4,[5],12:i:-) par réaction de polymérisation en Typhimurium (1,4,[5],12:i:-) durch Polymerase-
chaîne (PCR) (ISO 6579-4:2025) Kettenreaktion (PCR) (ISO 6579-4:2025)
This European Standard was approved by CEN on 27 January 2025.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 6579-4:2025 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 6579-4:2025) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by August 2025, and conflicting national standards shall
be withdrawn at the latest by August 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 6579-4:2025 has been approved by CEN as EN ISO 6579-4:2025 without any
modification.
International
Standard
ISO 6579-4
First edition
Microbiology of the food chain —
2025-01
Horizontal method for the
detection, enumeration and
serotyping of Salmonella —
Part 4:
Identification of monophasic
Salmonella Typhimurium
(1,4,[5],12:i:-) by polymerase
chain reaction (PCR)
Microbiologie de la chaîne alimentaire — Méthode horizontale
pour la recherche, le dénombrement et le sérotypage des
Salmonella —
Partie 4: Identification du variant monophasique de
Salmonella Typhimurium (1,4,[5],12:i:-) par réaction de
polymérisation en chaîne (PCR)
Reference number
ISO 6579-4:2025(en) © ISO 2025
ISO 6579-4:2025(en)
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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
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Email: copyright@iso.org
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Published in Switzerland
ii
ISO 6579-4:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 2
4 Principle . 2
4.1 General .2
4.2 Preparation of well-isolated colonies .2
4.3 Suspension of a colony .2
4.4 Amplification and detection .3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Presumptive monophasic Salmonella Typhimurium . 4
8 Culturing the isolate . 4
9 Procedure . 4
9.1 Preparation of cell suspension or DNA .4
9.2 PCR amplification and detection .4
9.2.1 General .4
9.2.2 PCR controls .4
10 Expression of results . 4
11 Performance characteristics of the method . 5
11.1 Validation in accordance with ISO 17468 .5
11.2 Performance characteristics .5
11.2.1 Method(s) evaluation study .5
11.2.2 Interlaboratory study .5
12 Test report . 6
13 Quality assurance . 6
Annex A (normative) Culture media and reagents. 7
Annex B (informative) Probe-based multiplex real-time PCR assay for the identification of
monophasic Salmonella Typhimurium (1,4,[5],12:i:-) . 9
Annex C (informative) Agarose gel-based multiplex target PCR assay for the identification of
monophasic Salmonella Typhimurium (1,4,[5],12:i:-) .13
Annex D (informative) Agarose gel-based single target PCR assay for the identification of
monophasic Salmonella Typhimurium (1,4,[5],12:i:-) .18
Annex E (informative) Performance characteristics .25
Bibliography .32
iii
ISO 6579-4:2025(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical Committee
CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical cooperation
between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 6579 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO 6579-4:2025(en)
Introduction
In several international, regional and national laws, regulatory limits are set to ensure the so-called
“absence” of Salmonella spp. in samples of the food chain. Moreover, several European Commission (EC)
regulations also demand the absence of particular Salmonella serovars which have shown to cause a
relatively high percentage of human salmonellosis. One of these Salmonella serovars for which legal criteria
are set is Salmonella Typhimurium, including its monophasic variant 1,4,[5],12:i:- (e.g. Regulation (EC)
[10]
No. 1086/2011 ). Hence, it is important to know that a serovar found with antigenic formula 1,4,[5],12:i:-
is indeed the monophasic variant of Salmonella Typhimurium (1,4,[5],12:i:1,2) and not the monophasic
variant of another Salmonella (S.) serovar for which no criteria are set, such as S. Lagos (1,4,[5],12:i:1,5),
S. Agama (4,12:i:1,6), S. Farsta (4,[5],12:i:e,n,x), S. Tsevie (1,4,12:i:e,n,z ), S. Gloucester (1,4,12,27:i:l,w) or
S. Tumodi (1,4,12:i:z ). Confirmational distinction between Salmonella Typhimurium and Salmonella non-
Typhimurium serovars can be determined using molecular analysis, such as the PCR technique(s) described
in this document.
v
International Standard ISO 6579-4:2025(en)
Microbiology of the food chain — Horizontal method for the
detection, enumeration and serotyping of Salmonella —
Part 4:
Identification of monophasic Salmonella Typhimurium
(1,4,[5],12:i:-) by polymerase chain reaction (PCR)
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detecting, enumerating and (sero)typing Salmonella are only undertaken in properly equipped
laboratories, under the control of a skilled microbiologist, and that great care is taken in the disposal
of all incubated materials. Persons using this document should be familiar with normal laboratory
practice. This document does not purport to address all of the safety aspects, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices.
1 Scope
This document specifies a horizontal in vitro method for the molecular identification and differentiation of
the monophasic variant of Salmonella enterica subsp. enterica serovar Typhimurium (1,4,[5],12:i:-) lacking
the second H phase H:1,2, starting from isolates. The method detects specific DNA sequences of an intergenic
region of the first H phase flagellin cluster for identification of Salmonella enterica subsp. enterica serovar
Typhimurium (further called Salmonella Typhimurium) and specific DNA sequences of genes associated
with second H phase flagellar antigen expression.
The method is applicable for:
— differentiation of the isolate under analysis between monophasic Salmonella Typhimurium and the
monophasic variant of another Salmonella non-Typhimurium serovar that has the same antigenic
formula;
— identification of the isolate under analysis being either monophasic Salmonella Typhimurium
or (biphasic) Salmonella Typhimurium.
This document is applicable for the analysis of a pure culture belonging to the genus Salmonella, isolated from:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.
This document can also be applied in other domains for identification of monophasic Salmonella Typhimurium
(e.g. environmental, human health, animal health).
NOTE This method has been validated in a method evaluation study and in an interlaboratory study with a large
set of different strains (target and non-target strains), isolated from different sources (food products, animals, animal
feed, primary production samples and humans). For detailed information on the validation, see Annex E.
ISO 6579-4:2025(en)
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 7218, Microbiology of the food chain — General requirements and guidance for microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance
testing of culture media
ISO 20836, Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of microorganisms
— Thermal performance testing of thermal cyclers
ISO 22174, Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection and quantification
of microorganisms — General requirements and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 22174 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
monophasic Salmonella Typhimurium
variant of Salmonella enterica subsp. enterica serovar Typhimurium lacking the second H phase H:1,2, having
the antigenic formula 1,4,[5],12:i:-
3.2
presumptive monophasic Salmonella Typhimurium
pure culture characterized as belonging to the genus Salmonella, giving a positive reaction for O-antigen O:4
and H-antigen H:i and with a negative reaction for the second H phase H:1,2
3.3
threshold cycle crossing point
point of the amplification curve at which the fluorescence signal rises above the baseline or crosses a
predefined threshold setting
4 Principle
4.1 General
The identification of the monophasic variant of Salmonella Typhimurium comprises the three successive
steps described in 4.2 to 4.4, starting with a pure culture characterized as belonging to the genus Salmonella.
4.2 Preparation of well-isolated colonies
The culture is streaked onto the surface of a (pre-dried) non-selective agar medium and incubated between
34 °C and 38 °C for 24 h, to obtain well-isolated colonies.
4.3 Suspension of a colony
A well-isolated colony is selected and suspended in 100 µl saline solution (0,85 % m/v) or in 100 µl PCR
grade water.
ISO 6579-4:2025(en)
4.4 Amplification and detection
The suspended bacterial cells are analysed by PCR for detection of the genetic sequences unique to Salmonella
Typhimurium (1,4,[5],12:i:1,2) and its monophasic variant lacking the second H phase (1,4,[5],12:i:-), as well
as for detection of specific genetic sequences of genes associated with the second H phase flagellar antigen
expression. Specific PCR assays including primers and probes are described in Annexes B to D.
5 Culture media and reagents
Follow current laboratory practices in accordance with ISO 7218. For the steps in 4.3 and 4.4, use reagents
and consumables of quality suitable for molecular biological applications (see ISO 22174). The composition
of culture media and reagents and their preparation are specified in Annex A. For performance testing of
culture media, follow the procedures in accordance with ISO 11133 and Clause A.4. The primers and probes for
identification of the monophasic variant of Salmonella Typhimurium (1,4,[5],12:i:-) are listed in Annexes B to D.
6 Equipment and consumables
Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications. The
usual microbiological laboratory equipment (see ISO 7218) and molecular biology equipment (see ISO 22174)
and, in particular, the following shall be used.
6.1 Incubator, capable of operating in the range of 34 °C to 38 °C.
NOTE The range 34 °C to 38 °C for incubation of culture media includes the use of incubators set at 35 °C ± 1 °C,
36 °C ± 2 °C or 37 °C ± 1 °C.
6.2 Sterile loops, of approximate diameter 3 mm (10 µl) or 0,3 mm (1 µl), or an inoculation needle/wire.
6.3 Water bath, capable of operating at 47 °C to 50 °C.
6.4 Refrigerator, capable of operating at 5 °C ± 3 °C.
6.5 Drying cabinet or oven, capable of operating between 25 °C and 50 °C.
6.6 pH-meter, having an accuracy of calibration of ± 0,1 pH unit at 20 °C to 25 °C.
6.7 Equipment for suspension of a colony, e.g. (micro)centrifuge tubes.
6.8 Graduated pipettes and pipette filter tips, for handling volumes between 0,2 µl and 13,55 µl,
depending on the PCR assay used (see Annex B, C or D). For more reactions per mix, larger volumes are needed.
6.9 Vortex mixer or equivalent.
6.10 Sterile Petri dishes, with a diameter of approximately 90 mm.
6.11 Equipment for PCR and real-time PCR, e.g. microcentrifuge or plate spinner.
6.12 Thermal cycler or real-time PCR thermal cycler, calibrated in accordance with ISO 20836.
6.13 Associated consumables for conventional or real-time PCR, e.g. PCR tubes, optical plates and seals,
optical plate holder, suitable for use with the selected PCR machine (see Annex B, C or D).
6.14 Apparatus for dry sterilization (oven) or wet sterilization (autoclave), as specified in ISO 7218.
ISO 6579-4:2025(en)
7 Presumptive monophasic Salmonella Typhimurium
The isolate to be used for further identification shall be a pure culture characterized as belonging to the
genus Salmonella (see ISO 6579-1). A presumptive monophasic Salmonella Typhimurium will show a
positive reaction for O-antigen O:4 and H-antigen H:i and a negative reaction for the second H phase (see
ISO/TR 6579-3).
8 Culturing the isolate
Streak the culture of Clause 7 (e.g. with a 10 µl loop; 6.2) on the surface of a non-selective agar medium (e.g.
nutrient agar; Clause A.2) to obtain well-isolated colonies. Incubate the plates, inverted, between 34 °C and
38 °C (6.1) for 24 h ± 3 h.
9 Procedure
9.1 Preparation of cell suspension or DNA
By means of an inoculating wire or a sterile loop (6.2), pick and suspend (a portion of) one colony in 100 µl
saline solution (0,85 % m/v; Clause A.3) or in 100 µl PCR grade water in an appropriate tube (6.7).
Mix (6.9) for homogenization of the suspension.
An aliquot of 2 µl or 2,5 µl, depending on the specific PCR assay, of this bacterial cell suspension is used (see
Table B.2, C.2, D.2, D.3 or D.4).
It is also possible to use a DNA extract for the PCR assay. For DNA extraction, for example, thermal cell lysis
can be used, or another appropriate extraction method. If shown to be reliable, commercial kits can also be
used for DNA extraction, following the manufacturer’s instructions.
9.2 PCR amplification and detection
9.2.1 General
Different protocols for multiplex probe-based real-time PCR, multiplex PCR followed by agarose gel
electrophoresis detection of the amplification products or single target PCR followed by agarose gel
electrophoresis detection can be used.
A probe-based multiplex real-time PCR assay is given in Annex B.
An agarose gel-based multiplex PCR assay is given in Annex C.
An agarose gel-based single target PCR assay is given in Annex D.
Follow all requirements, including the use of suitable equipment (6.11), for the (real-time) PCR amplification
as specified in ISO 22174.
9.2.2 PCR controls
Use (process) controls for the PCR assays in accordance with ISO 22174.
For the real-time PCR (see Annex B) and the single target PCR (see Annex D), an internal amplification
control (IAC) shall also be used as the targets could all be negative. Since the multiplex PCR (see Annex C)
will always result in a PCR fragment (1 000 bp or 250 bp), this procedure does not require an IAC.
10 Expression of results
The results obtained, including controls specified in ISO 22174, shall be unambiguous otherwise the PCR
shall be repeated.
ISO 6579-4:2025(en)
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 controls give
expected results.
If the PCR assay identifies the isolate as monophasic Salmonella Typhimurium, report the result preferably
by giving the antigenic formula as determined.
It is possible that an isolate is phenotypically identified as monophasic Salmonella Typhimurium, but
genotypically (with PCR) as biphasic Salmonella Typhimurium. This can be caused by the fact that the genes
are present, but phenotypically not expressed. For identification of these isolates, the PCR results take
precedence over serum agglutination test results.
11 Performance characteristics of the method
11.1 Validation in accordance with ISO 17468
The PCR methods described in Annexes B, C and D were validated in accordance with ISO 17468. All relevant
data as obtained in steps 1 to 5 of ISO 17468, as well as the results of the interlaboratory study (step 6 in
ISO 17468) were reported in Reference [8].
The performance characteristics of the three PCR methods (inclusivity and exclusivity) were determined in
a method(s) evaluation study (described in 11.2.1) and in an interlaboratory study (described in 11.2.2).
11.2 Performance characteristics
11.2.1 Method(s) evaluation study
The three PCR assays described in Annexes B, C and D were tested in a method evaluation study, by analysing
172 different strains (target and non-target strains), isolated from different sources (food products, animals,
animal feed, primary production samples and humans), in two different laboratories. For the inclusivity and
the exclusivity testing, the typing results of Salmonella found by slide agglutination were compared to the
typing results found by each PCR method.
All data are given in Annex E and more details can be found in Reference [8].
It depends on the intended specific purpose for which the PCR assay is being applied and its performance
evaluated, as to whether only monophasic Salmonella Typhimurium is considered as target strain (and thus
part of the inclusivity study) or if (biphasic) Salmonella Typhimurium is also considered as target strain.
If the intended purpose is to determine if the strain under analysis is the monophasic variant of Salmonella
Typhimurium and not the monophasic variant of another Salmonella non-Typhimurium serovar, then
monophasic Salmonella Typhimurium as well as (biphasic) Salmonella Typhimurium can be considered
as target strains and the three PCR assays described in Annexes B, C and D perform equally well for
identification of monophasic Salmonella Typhimurium strains (see Table E.1).
If the aim is to identify whether the strain under analysis is either monophasic Salmonella Typhimurium
or (biphasic) Salmonella Typhimurium, then Salmonella Typhimurium should be considered as non-target
strain. For this purpose, the gel-based multiplex PCR (see Annex C) can be less specific for some strains
than the other two PCR assays (see Table E.2), as this assay is less suitable to distinguish biphasic from
monophasic Salmonella Typhimurium.
11.2.2 Interlaboratory study
The performance characteristics of each PCR method (see Annexes B, C and D) were determined in an
interlaboratory study (step 6 in ISO 17468) to determine the inclusivity and exclusivity of the three methods,
ISO 6579-4:2025(en)
following the procedures described in ISO 16140-6. Details about the interlaboratory study and a summary
of the data are given in Clause E.2 for each PCR assay.
A summary of the inclusivity and exclusivity data is given in Table E.4.
In the inclusivity study, pure target strains to be detected by the method were tested. For this interlaboratory
study, monophasic Salmonella Typhimurium was considered as the only target strain.
In the exclusivity study, pure non-target strains that are not expected to be detected by the method but
can potentially be cross-reactive were tested. For this interlaboratory study, Salmonella serovars other
than monophasic Salmonella Typhimurium, including (biphasic) Salmonella Typhimurium and other
Enterobacteriaceae were considered as non-target strains.
12 Test report
The test report shall specify at least the following:
— the test method used, with reference to this document, i.e. ISO 6579-4;
— all operating conditions not specified in this document, or regarded as optional or informative (including
informative annexes), together with details of any incidents which can have influenced the test result(s);
— any deviations from this document;
— all information necessary for the complete identification of the sample;
— the test result(s) obtained;
— the date of the test.
13 Quality assurance
The laboratory should have a quality control system to ensure that the equipment, reagents and techniques
are suitable for the method. The use of positive controls, negative controls and blanks are part of the method.
Performance testing of culture media is specified in Clause A.4 and described in ISO 11133.
ISO 6579-4:2025(en)
Annex A
(normative)
Culture media and reagents
A.1 General
The general specifications of ISO 11133 are applicable to the preparation and performance testing of the
culture media described in this annex. If culture media or reagents are prepared from dehydrated complete
media/reagents, or if ready-to-use media/reagents are used, follow the manufacturer’s instructions
regarding preparation, storage conditions, expiry date and use.
The shelf life of the media indicated in this annex has been shown in some studies. The user shall verify this
under their own storage conditions (as specified in ISO 11133).
Performance testing of culture media is described in Clause A.4.
A.2 Nutrient agar (example of non-selective agar medium)
A.2.1 Composition
Meat extract 3,0 g
a
Peptone 5,0 g
c
Sodium chloride (NaCl) (optional) (CAS Registry Number® 7647-14-5) 5,0 g
b
Agar 9,0 g to 18,0 g
Water 1 000 ml
a
For example, enzymatic digest of casein.
b
Depending on the gel strength of the agar.
c
Chemical Abstracts Service (CAS) Registry Number® is a trademark of the American Chemical Society
(ACS). This information is given for the convenience of users of this document and does not constitute an
endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to
the same results.
A.2.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by heating, with frequent
agitation.
Adjust the pH (6.6), if necessary, so that after sterilization, it is 7,0 ± 0,2 at 20 °C to 25 °C.
Transfer the culture medium into tubes or flasks of appropriate capacity.
Sterilize for 15 min in the autoclave (6.14) set at 121 °C.
A.2.3 Preparation of nutrient agar plates
Cool the medium to 47 °C to 50 °C in a water bath (6.3), mix, and pour into sterile Petri dishes up to a volume
of approximately 15 ml to 20 ml in dishes with a diameter of approximately 90 mm (6.10). Allow to solidify.
Immediately before use, dry the agar plates carefully (preferably with the lids off and the agar surface
downwards) in a drying cabinet or oven set between 25 °C and 50 °C (6.5) until the surface of the agar is dry.
Store the poured plates protected from drying, at 5 °C (6.4) for up to four weeks.
ISO 6579-4:2025(en)
A.3 Saline solution (0,85 % m/v)
A.3.1 Composition ®
Sodium chloride (NaCl) (CAS RN 7647-14-5) 8,5 g
Water 1 000 ml
A.3.2 Preparation
Dissolve the sodium chloride in the water.
Adjust the pH (6.6), if necessary, so that after sterilization, it is 7,0 ± 0,2 at 20 °C to 25 °C.
Dispense the solution into flasks or tubes of suitable capacity to obtain the portions necessary for the test.
Sterilize for 15 min in the autoclave (6.14) set at 121 °C.
Store the solution in closed flasks or tubes at 5 °C (6.4) for up to six months.
A.4 Performance testing for the quality assurance of the culture media
The definition of selectivity and productivity is specified in ISO 11133. In general, follow the procedures for
performance testing described in ISO 11133, including the inoculum levels for the target and the non-target
organisms. Table A.1 gives details of control strains to be used for performance testing of culture media
specified in this document.
Table A.1 — Performance testing for the quality assurance of the culture media
a b
Medium Function Incubation Control strains WDCM numbers Criteria
c,d
Nutrient Productivity 24 h ± 3 h/ Salmonella Typhimurium 00031 Good growth
agar 34 °C to 38 °C
c,d
Salmonella Enteritidis 00030
a
Refer to the reference strain catalogue at www .wfcc .info for information on culture collection strain numbers and contact
details; WDCM: World Data Centre for Microorganisms.
b
Growth is categorized as 0: no growth, 1: weak growth (partial inhibition), and 2: good growth (see ISO 11133).
c
Some national restrictions and directions can require the use of a different serovar. Make reference to national requirements
relating to the choice of Salmonella serovars.
d
Strain free of choice; one of the strains shall be used as a minimum.
ISO 6579-4:2025(en)
Annex B
(informative)
Probe-based multiplex real-time PCR assay for the identification of
monophasic Salmonella Typhimurium (1,4,[5],12:i:-)
B.1 General
This annex describes a probe-based multiplex real-time PCR method based on 5′- nuclease technology for the
identification of the monophasic variant of Salmonella Typhimurium (1,4,[5],12:i:-) and the differentiation
from other Salmonella non-Typhimurium monophasic serovars, by targeting the following genetic sequences:
— linkage between fliA and insertion sequence IS200 (present in Salmonella Typhimurium (1,4,[5],12:i:1,2)
and in monophasic Salmonella Typhimurium (1,4,[5],12:i:-));
— linkage between fljB and hin (present in isolates expressing the second H phase antigen);
— linkage between hin and iroB (present in isolates expressing the second H phase antigen).
For the expression of the second H phase antigen, both amplification products, fljB – hin and hin – iroB,
shall be detected. If one of the two products is not detected, expression of the second H phase antigen is
interrupted.
The probe-based multiplex real-time PCR also contains a heterologous internal amplification control (IAC)
based on the plasmid pUC18/19. By real-time PCR, a fragment spanning from M13pm18 to sequences of
pBR322 is amplified. This sequence does not occur naturally.
NOTE Another IAC, with its specific primers and probe, can be used if it produces equivalent results.
B.2 Procedure
B.2.1 Principle
Three specific genetic sequences and a sequence of the IAC are amplified and detected by a probe-based
multiplex real-time PCR method based on 5′- nuclease technology.
B.2.2 Reagents for PCR
B.2.2.1 General
See ISO 22174.
B.2.2.2 Primers and probes
Primers and probes are published in Reference [7]. The sequences are listed in Table B.1.
ISO 6579-4:2025(en)
Table B.1 — Sequences of the primers and probes
Target Primer/probe Primer/probe sequence (5′-3′) Amplicon
sequence name size (bp)
fliA-IS200F CAT TAC ACC TTC AGC GGT AT
fliA-IS200 fliA-IS200R CTG GTA AGA GAG CCT TAT AGG 254
a
fliA-IS200-probe2 FAM – CGG CAT GAT TAT CCG TTT CTA CAG AGG – BHQ1
fljB-hinF TGG TGC TGT TAG CAG AC
fljB-hin fljB-hinR TCA ACA CTA ACA GTC TGT CG 297
b
fljB-hin-probe YY – AAC CGC CAG TTC ACG CAC – BHQ2
hin-iroBF GTG TGG CAT AAA TAA ACC GA
hin-iroB hin-iroBR AGG CTT ACC TGT GTC ATC CA 274
c
hin-iroB-probe ROX – TAA CGC GCT CAC GAT AAG GC – BHQ2
IAC-FW GTC GGG AAA CCT GTC G
d
IAC IAC-RV GCT CAC ATG TTC TTT CCT GC 207
e
IAC-probe Cy5 – CGG GGA GAG GCG GTT– BHQ3
NOTE Other appropriate fluorophores and quenchers can be used.
a f
FAM: 6 Carboxyfluorescein, BHQ1: Black Hole Quencher 1™.
b f
YY: Yakima Yellow™, BHQ2: Black Hole Quencher 2™.
c f
ROX: Carboxy-X-rhodamine™, BHQ2: Black Hole Quencher 2™.
d
IAC: Internal amplification control.
e ® f
Cy5: Cyanine 5 , BHQ3: Black Hole Quencher 3™.
f
Products with trade names or trademarks are given for the convenience of users of this document and does not constitute an
endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.
B.2.3 Real-time PCR setup
B.2.3.1 Reaction setup
The total PCR volume is 25 µl per PCR reaction. The reagents for preparation of the reaction mix are listed
in Table B.2.
Table B.2 — Preparation of the reaction mix
Reagent Final concentration Volume per reaction
a
(stock concentration) (µl)
10× PCR buffer containing no magnesium chloride 1× 2,5
dNTP-mix (each 2 mmol/l) Each 200 µmol/l 2,5
Magnesium chloride (50 mmol/l) 2,5 mmol/l 1,25 ®
(CAS RN 7786-30-3)
fliA-IS200F (20 pmol/µl) 0,4 pmol/µl 0,5
fliA-IS200R (20 pmol/µl) 0,4 pmol/µl 0,5
fljB-hinF (20 pmol/µl) 0,4 pmol/µl 0,5
fljB-hinR (20 pmol/µl) 0,4 pmol/µl 0,5
hin-iroBF (20 pmol/µl) 0,4 pmol/µl 0,5
NOTE For IAC, pUC 18 can also be used. 1 fg pUC 18/19 corresponds to approximately 3,4 × 10 copies.
a
Volume per reaction depends on the concentration of the reagent in the stock solution and is determined by the final
concentration.
b
Performance characteristics of this assay were tested with the commercially available Platinum™ Taq Polymerase and
10× PCR buffer (Invitrogen). This information is given for the convenience of the user of this document and does not constitute an
endorsement by ISO of this product. Equivalent products may be used if they can be shown to produce equivalent results.
ISO 6579-4:2025(en)
TTabablele B B.22 ((ccoonnttiinnueuedd))
Reagent Final concentration Volume per reaction
a
(stock concentration) (µl)
hin-iroBR (20 pmol/µl) 0,4 pmol/µl 0,5
IAC-FW (20 pmol/µl) 0,4 pmol/µl 0,5
IAC-RV (20 pmol/µl) 0,4 pmol/µl 0,5
fliA-IS200-probe2 (5′FAM-3′BHQ1) (5 pmol/µl) 0,125 pmol/µl 0,625
fljB-hin-probe (5′YY-3′BHQ2) (10 pmol/µl) 0,25 pmol/µl 0,625
hin-iroB-probe (5′ROX-3′BHQ2) (10 pmol/µl) 0,25 pmol/µl 0,625
IAC-probe (5′Cy5–3′BHQ3) (10 pmol/µl) 0,25 pmol/µl 0,625
b
Taq Polymerase 2 U 0,2
IAC-pUC19DNA Approximately 10 copies 1,0
...
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