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, zur Zählung und zur Serotypisierung von Salmonellen - 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 In-vitro-Verfahren für die molekulare Identifizierung und Differenzierung der monophasischen Variante von Salmonella enterica subsp. enterica serovar Typhimurium (1,4[5],12:i: ), bei der die zweite H-Phase H:1,2 fehlt, und mit den Isolaten beginnt fest. Dieses Verfahren weist spezifische DNA-Sequenzen einer intergenischen Region der ersten H-Phase des Flagellen-Clusters nach, um Salmonella enterica subsp. enterica serovar Typhimurium (im folgenden Salmonella Typhimurium) sowie spezifische DNA-Sequenzen von Genen, die mit der zweiten H-Phase der Flagellen-Antigen-Expression verbunden sind, zu identifizieren.
Das Verfahren ist anwendbar zur:
Differenzierung des untersuchten Isolats zwischen monophasischem Salmonella Typhimurium und der monophasischen Variante eines anderen Salmonella non Typhimurium-Serovars, das die gleiche Antigenformel hat;
Identifizierung des untersuchten Isolats, das entweder ein monophasisches Salmonella Typhimurium oder ein (biphasisches) Salmonella Typhimurium ist.
Dieses Dokument ist anwendbar auf die Untersuchung einer zur Gattung Salmonellen gehörenden Reinkultur, welche isoliert wurde von:
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.
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/FDIS 6579-4:2024)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN ISO 6579-4:2024
01-marec-2024
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
prisotnosti, števila in serotipov Salmonella - 4. del: Identifikacija monofazne
Salmonelle Typhimurium (1,4,[5],12:i:-) z verižno reakcijo s polimerazo (PCR)
(ISO/DIS 6579-4:2023)
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/DIS 6579-4:2023)
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/DIS 6579-4:2023)
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/DIS 6579-4:2023)
Ta slovenski standard je istoveten z: prEN ISO 6579-4
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 6579-4:2024 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
oSIST prEN ISO 6579-4:2024
oSIST prEN ISO 6579-4:2024
DRAFT INTERNATIONAL STANDARD
ISO/DIS 6579-4
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2023-12-22 2024-03-15
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)
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche, le dénombrement et le
sérotypage des Salmonella —
Partie 4: Identification de Salmonella Typhimurium (1,4,[5],12:i:-) monophasique par réaction de
polymérisation en chaîne (PCR)
ICS: 07.100.30
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oSIST prEN ISO 6579-4:2024
ISO/DIS 6579-4:2023(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 6579-4
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
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)
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche, le dénombrement et le
sérotypage des Salmonella —
Partie 4: Identification de Salmonella Typhimurium (1,4,[5],12:i:-) monophasique par réaction de
polymérisation en chaîne (PCR)
ICS: 07.100.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2023
ISO/CEN PARALLEL PROCESSING
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ii
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023
oSIST prEN ISO 6579-4:2024
ISO/DIS 6579-4:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
4.1 General . 3
4.2 Preparation of well-isolated colonies . 3
4.3 Suspension of a colony . 3
4.4 Amplification and detection . 3
5 Culture media and reagents .3
6 Equipment and consumables .3
7 Presumptive monophasic Salmonella Typhimurium strain (isolate) .4
8 Culturing the isolate .4
9 Procedure .4
9.1 Preparation of cell suspension or DNA . 4
9.2 PCR amplification and detection . 5
9.2.1 General . 5
9.2.2 PCR controls . 5
10 Expression of results . 5
11 Performance characteristics of the method . 6
11.1 Validation in accordance with ISO 17468 . 6
11.2 Performance characteristics . 6
11.2.1 Method(s) evaluation study . 6
11.2.2 Interlaboratory study . 6
12 Test report . 7
13 Quality assurance . 7
Annex A (informative) Culture media and reagents . 8
Annex B (informative) Probe-based multiplex real-time PCR assay for the identification of
monophasic Salmonella Typhimurium (1,4[5],12:i:-) .10
Annex C (informative) Agarose gel-based multiplex target PCR assay for the identification
of monophasic Salmonella Typhimurium (1,4[5],12:i:-) .14
Annex D (informative) Agarose gel-based single target PCR assay for the identification of
monophasic Salmonella Typhimurium (1,4[5],12:i:-) .19
Annex E (informative) Performance characteristics .26
Bibliography .32
iii
oSIST prEN ISO 6579-4:2024
ISO/DIS 6579-4:2023(E)
Foreword
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different types of ISO documents should be noted. This document was drafted in accordance with the
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This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
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.
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iv
oSIST prEN ISO 6579-4:2024
ISO/DIS 6579-4:2023(E)
Introduction
In several (international, regional, national) laws, regulatory limits are set to ensure the ‘absence’
of Salmonella spp. in samples of the food chain. Moreover, several (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],
...
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