Microbiology of the food chain - Method validation - Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing procedures (ISO 16140-6:2019)

This document specifies the general principle and the technical protocol for the validation of alternative confirmation methods for microbiology in the food chain. This document compares the result of the alternative confirmation method against the confirmation procedure of a reference method or, if needed, a reference confirmation method (e.g. whole genome sequencing).
This document is applicable to the validation of alternative confirmation methods used for the analysis (detection or quantification) of isolated microorganisms in:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production, handling;
— samples from the primary production stage.
Validated alternative confirmation methods can be used to replace (partly or completely) the confirmation procedure described in:
— the reference method;
— an alternative method validated in accordance with ISO 16140-2 only if one of the isolation agars specified in the validation study of the alternative confirmation method is used.
This document is also applicable to the validation of alternative typing methods, where the reference method can be, for example, a serological method (e.g. serotyping of Salmonella) or a molecular method (e.g. typing of Shiga toxin-producing E. coli).
This document is, in particular, applicable to bacteria and fungi. Some clauses can be applicable to other (micro)organisms, to be determined on a case-by-case basis.
Validation studies in accordance with this document are primarily intended to be performed by organizations or expert laboratories involved in method validation, but can also be used by a single laboratory, especially when performing in-house validation under certain conditions (see ISO 16140-4).

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 6: Arbeitsvorschrift für die Validierung von (urheberrechtlich geschützten) Alternativverfahren für die Bestätigungs- und Typisierungsprüfung (ISO 16140-6:2019)

Dieses Dokument legt das allgemeine Prinzip und die technische Arbeitsvorschrift für die Validierung alternativer Bestätigungsverfahren in der Mikrobiologie der Lebensmittelkette fest. Dieses Dokument vergleicht die Ergebnisse des alternativen Bestätigungsverfahrens mit denen des Bestätigungsvorgangs eines Referenzverfahrens oder, falls erforderlich, eines Referenzbestätigungsverfahrens (z. B. Gesamtgenom¬sequenzierung).
Dieses Dokument ist anwendbar auf die Validierung von alternativen Bestätigungsverfahren zur Analyse (Nachweis oder quantitative Bestimmung) von isolierten Mikroorganismen in:
   Erzeugnissen, die für den menschlichen Verzehr vorgesehen sind;
   Erzeugnissen, die als Futtermittel vorgesehen sind;
   Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln;
   Proben aus dem Bereich der Primärproduktion.
Validierte alternative Bestätigungsverfahren können verwendet werden, um das Bestätigungsverfahren (ganz oder teilweise) zu ersetzen, das beschrieben ist
   im Referenzverfahren,
   in einem Alternativverfahren, das nach ISO 16140 2 validiert worden ist, sofern dabei eines der in der Validierungsuntersuchung des alternativen Bestätigungsverfahrens angegebenen Isolierungs Agars verwendet wird.
Darüber hinaus ist dieses Dokument auch anwendbar auf die Validierung von alternativen Typisierungsverfahren, bei denen das Referenzverfahren beispielsweise ein serologisches Verfahren (z. B. zur Serotypisierung von Salmonella) oder ein molekulares Verfahren ist (z. B. zur Typisierung von Shigatoxin bildenden E. coli).
Dieses Dokument gilt insbesondere für Bakterien und Pilze. Einige Abschnitte sind auch auf andere (Mikro )Organismen anwendbar. Die Bestimmung muss im Einzelfall erfolgen.
Validierungsuntersuchungen nach diesem Dokument sollen in erster Linie von Organisationen oder Expertenlaboren durchgeführt werden, die an der Verfahrensvalidierung beteiligt sind. Sie können aber auch von einem Einzel-Labor verwendet werden, insbesondere bei der laborinternen Validierung unter bestimmten Bedingungen (siehe ISO 16140 4).

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 6: Protocole pour la validation de méthodes alternatives (commerciales) pour la confirmation microbiologique et le typage (ISO 16140-6:2019)

Le présent document établit le principe général ainsi que le protocole technique pour la validation des méthodes alternatives de confirmation dans le domaine de la microbiologie de la chaîne alimentaire. Le présent document compare le résultat de la méthode alternative de confirmation avec celui du mode opératoire de confirmation d'une méthode de référence ou, si nécessaire, d'une méthode de confirmation de référence (par exemple, séquençage de génome complet).
Le présent document est applicable à la validation de méthodes alternatives de confirmation utilisées pour l'analyse (recherche ou quantification) de micro-organismes isolés présents dans:
— les produits destinés à la consommation humaine;
— les produits destinés à l'alimentation animale;
— les échantillons environnementaux dans les domaines de la production et de la manutention de produits alimentaires;
— les échantillons au stade de la production primaire.
Des méthodes alternatives de confirmation validées peuvent être utilisées pour remplacer (en partie ou en totalité) le mode opératoire de confirmation décrit dans:
— la méthode de référence;
— une méthode alternative validée conformément à l'ISO 16140‑2 uniquement si l'une des géloses d'isolement spécifiées dans l'étude de validation de la méthode de confirmation alternative est utilisée.
Le présent document est également applicable à la validation de méthodes alternatives de typages, dans lesquelles la méthode de référence peut, par exemple, être une méthode sérologique (par exemple, sérotypage de Salmonella) ou une méthode moléculaire (par exemple, typage d'E. coli produisant des shigatoxines).
Le présent document est notamment applicable aux bactéries et aux champignons. Certains articles peuvent être applicables à d'autres (micro)organismes, qui doivent être déterminés au cas par cas.
Les études de validation conformément au présent document sont principalement destinées à être effectuées par des organismes ou des laboratoires experts impliqués dans la validation des méthodes, mais peuvent également être utilisées par un seul laboratoire, notamment lors de la réalisation d'une validation interne sous certaines conditions (voir ISO 16140‑4).

Mikrobiologija v prehranski verigi - Validacija metode - 6. del: Protokol za validacijo alternativnih (lastniških) metod za postopke mikrobiološke potrditve in tipizacije (ISO 16140-6:2019)

Ta del standarda ISO 16140 določa splošno načelo in tehnični protokol za validacijo alternativnih, večinoma lastniških metod za potrditev mikrobiološke analize hrane, krme za živali ter vzorcev okoljske in primarne proizvodnje. Ta postopek je omejen na validacijo alternativnih (lastniških) metod za potrditev, ki naj bi delno ali v celoti nadomestile postopek potrditve, opisan v standardni metodi štetja ali zaznavanja specifičnih (skupin) mikroorganizmov. »Vzorec« za potrditev je sumljiva kolonija, ki je bila pridobljena s postopkom za pridobivanje referenčne ali alternativne kulture. Postopek ni namenjen potrditvi z uporabo (čiste) kolonije neznanega izvora. Validativne študije v skladu s tem standardom naj bi izvajale organizacije, ki sodelujejo pri validaciji metod.

General Information

Status
Published
Publication Date
03-Dec-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
04-Dec-2019
Completion Date
04-Dec-2019

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SLOVENSKI STANDARD
SIST EN ISO 16140-6:2020
01-februar-2020
Mikrobiologija v prehranski verigi - Validacija metode - 6. del: Protokol za

validacijo alternativnih (lastniških) metod za postopke mikrobiološke potrditve in

tipizacije (ISO 16140-6:2019)

Microbiology of the food chain - Method validation - Part 6: Protocol for the validation of

alternative (proprietary) methods for microbiological confirmation and typing procedures

(ISO 16140-6:2019)

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 6: Arbeitsvorschrift für

die Validierung (urheberrechtlich geschützter) Alternativverfahren für die Bestätigungs-

und Typisierungsprüfung (ISO 16140-6:2019)

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 6: Protocole

pour la validation de méthodes alternatives (commerciales) pour la confirmation
microbiologique et le typage (ISO 16140-6:2019)
Ta slovenski standard je istoveten z: EN ISO 16140-6:2019
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-6:2020 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16140-6:2020
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SIST EN ISO 16140-6:2020
EN ISO 16140-6
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2019
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Method validation - Part 6:
Protocol for the validation of alternative (proprietary)
methods for microbiological confirmation and typing
procedures (ISO 16140-6:2019)

Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -

méthodes - Partie 6: Protocole pour la validation de Verfahrensvalidierung - Teil 6: Arbeitsvorschrift für die

méthodes alternatives (commerciales) pour la Validierung (urheberrechtlich geschützter)

confirmation microbiologique et le typage (ISO 16140- Alternativverfahren für die Bestätigungs- und

6:2019) Typisierungsprüfung (ISO 16140-6:2019)
This European Standard was approved by CEN on 10 September 2019.

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, Turkey 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

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-6:2019 E

worldwide for CEN national Members.
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SIST EN ISO 16140-6:2020
EN ISO 16140-6:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 16140-6:2020
EN ISO 16140-6:2019 (E)
European foreword

This document (EN ISO 16140-6:2019) 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 June 2020, and conflicting national standards shall be

withdrawn at the latest by June 2020.

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.

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, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 16140-6:2019 has been approved by CEN as EN ISO 16140-6:2019 without any

modification.
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SIST EN ISO 16140-6:2020
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SIST EN ISO 16140-6:2020
INTERNATIONAL ISO
STANDARD 16140-6
First edition
2019-11
Microbiology of the food chain —
Method validation —
Part 6:
Protocol for the validation of
alternative (proprietary) methods
for microbiological confirmation and
typing procedures
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 6: Protocole pour la validation de méthodes alternatives
(commerciales) pour la confirmation microbiologique et le typage
Reference number
ISO 16140-6:2019(E)
ISO 2019
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SIST EN ISO 16140-6:2020
ISO 16140-6:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

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 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 16140-6:2020
ISO 16140-6:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 2

4 General principles for the validation of confirmation and typing methods...........................................3

5 Strains .............................................................................................................................................................................................................................. 4

6 Method comparison study .......................................................................................................................................................................... 4

6.1 General ........................................................................................................................................................................................................... 4

6.2 Selection of test strains .................................................................................................................................................................... 4

6.3 Inclusivity study ..................................................................................................................................................................................... 4

6.3.1 Testing of target strains ............................................................................................................................................. 4

6.3.2 Family level (non-Salmonella) .............................................................................................................................. 5

6.3.3 Genus level (non-Salmonella) ............................................................................................................................... 5

6.3.4 Species level (non-Salmonella) ............................................................................................................................ 5

6.3.5 Microbial (sub)type level (non-Salmonella) ............................................................................................ 5

6.3.6 Salmonella genus or species level ..................................................................................................................... 5

6.3.7 Salmonella serovar level ............................................................................................................................................ 6

6.4 Exclusivity study .................................................................................................................................................................................... 6

6.4.1 Testing of non-target strains ................................................................................................................................. 6

6.4.2 Family level (non-Salmonella) .............................................................................................................................. 6

6.4.3 Genus level (non-Salmonella) ............................................................................................................................... 6

6.4.4 Species level (non-Salmonella) ............................................................................................................................ 6

6.4.5 Microbial (sub)type level (non-Salmonella) ............................................................................................ 6

6.4.6 Salmonella genus or species level ..................................................................................................................... 7

6.4.7 Salmonella serovar level ............................................................................................................................................ 7

6.5 Expression and interpretation of results ......................................................................................................................... 7

6.6 Evaluation .................................................................................................................................................................................................... 9

7 Interlaboratory study ...................................................................................................................................................................................10

7.1 General ........................................................................................................................................................................................................10

7.2 Data sets to be obtained ...............................................................................................................................................................10

7.3 Protocol ......................................................................................................................................................................................................11

7.4 Expression of results .......................................................................................................................................................................12

7.5 Interpretation and evaluation .................................................................................................................................................12

Annex A (normative) Points to be considered when selecting strains for testing inclusivity

and exclusivity .....................................................................................................................................................................................................14

Annex B (informative) Example of the validation of an alternative confirmation method to

the species level (Listeria monocytogenes) ............................................................................................................................16

Annex C (informative) Example of the validation of an alternative typing method to the

Salmonella serovar level (15 different serovars claimed) .....................................................................................19

Annex D (normative) Overview of the various options for validation under this document

and the acceptability limits ....................................................................................................................................................................23

Bibliography .............................................................................................................................................................................................................................25

© ISO 2019 – All rights reserved iii
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SIST EN ISO 16140-6:2020
ISO 16140-6:2019(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 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.
A list of all parts of the ISO 16140 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 2019 – All rights reserved
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SIST EN ISO 16140-6:2020
ISO 16140-6:2019(E)
Introduction
0.1 The ISO 16140 series

The ISO 16140 series has been expanded in response to the need for various ways to validate or verify

test methods. It is the successor to ISO 16140:2003. The ISO 16140 series consists of six parts with the

general title, Microbiology of the food chain — Method validation:
— Part 1: Vocabulary;

— Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method;

— Part 3: Protocol for the verification of validated reference methods and validated alternative methods

in a single laboratory;
— Part 4: Protocol for method validation in a single laboratory;

— Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods;

— Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation

and typing procedures.

ISO 17468 is a closely linked International Standard, which establishes technical rules for the

development and validation of standardized methods.
In general, two stages are needed before a method can be used in a laboratory.

— The first stage is the validation of the method. Validation is conducted using a study in a single

laboratory followed by an interlaboratory study (see ISO 16140-2, ISO 16140-5 and as described in

this document). In the case when a method is validated within one laboratory (see ISO 16140-4), no

interlaboratory study is conducted.

— The second stage is method verification, where a laboratory demonstrates that it can satisfactorily

perform a validated method. This is described in ISO 16140-3. Verification is only applicable to

methods that have been validated using an interlaboratory study.

In general, two types of methods are distinguished: reference methods and alternative methods.

A reference method is defined in ISO 16140-1:2016, 2.59, as an “internationally recognized and widely

accepted method”. The note to entry clarifies that “these are ISO standards and standards jointly

published by ISO and CEN or other regional/national standards of equivalent standing”.

In the ISO 16140 series, reference methods include standardized reference (ISO and CEN) methods as

defined in ISO 17468:2016, 3.5, as a “reference method described in a standard”.

An alternative method (method submitted for validation) is defined in ISO 16140-1:2016, 2.4, as a

“method of analysis that detects or quantifies, for a given category of products, the same analyte as

is detected or quantified using the corresponding reference method”. The note to entry clarifies that:

“The method can be proprietary. The term ‘alternative’ is used to refer to the entire ‘test procedure

and reaction system’. This term includes all ingredients, whether material or otherwise, required for

implementing the method.”.

ISO 16140-4 addresses validation within a single laboratory. The results are therefore only valid for

the laboratory that conducted the study. In this case, verification (as described in ISO 16140-3) is not

applicable. ISO 16140-5 describes protocols for non-proprietary methods where a more rapid validation

is required or when the method to be validated is highly specialized and the number of participating

laboratories required by ISO 16140-2 cannot be reached. ISO 16140-4 and ISO 16140-5 can be used

for validation against a reference method. ISO 16140-4 (qualitative and quantitative) and ISO 16140-5

(quantitative only) can also be used for validation without a reference method.
© ISO 2019 – All rights reserved v
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SIST EN ISO 16140-6:2020
ISO 16140-6:2019(E)

The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above.

It also guides the user in selecting the right part of the ISO 16140 series, taking into account the purpose

of the study and the remarks given above.
Figure 1 — Flow chart for application of the ISO 16140 series

NOTE In this document, the words “category”, “type” and/or “item” are sometimes combined with “(food)”

to improve readability. However, the word “food” is interchangeable with “feed” and other areas of the food chain

as mentioned in Clause 1.

This document, ISO 16140-6, is somewhat different from the other parts in the ISO 16140 series in that it

relates to a very specific situation where only the confirmation procedure of a method is to be validated

[e.g. the biochemical confirmation of Enterobacteriaceae (see ISO 21528-2)]. The confirmation procedure

advances a suspected (presumptive) result to a confirmed positive result. The validation of alternative

typing techniques (e.g. serotyping of Salmonella) is also covered by this document. The validation study

in this document clearly defines the selective agar(s) from which strains can be confirmed using the

alternative confirmation method. If successfully validated, the alternative confirmation method can

only be used if strains are recovered on an agar that was used and shown to be acceptable within the

validation study. Figure 2 shows the possibilities where an alternative confirmation method validated

in accordance with this document can be applied (see text in the boxes).
vi © ISO 2019 – All rights reserved
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SIST EN ISO 16140-6:2020
ISO 16140-6:2019(E)

Figure 2 — Use of validated alternative confirmation methods (described in this document)

EXAMPLE An example application of a validated alternative confirmation method is as follows.

An alternative confirmation method based on ELISA has been validated to replace the biochemical confirmation

for Salmonella as described in ISO 6579-1. In the validation study, XLD (mandatory agar in accordance with

ISO 6579-1) plus BGA and a specified chromogenic agar (two optional agars for second plating in accordance with

ISO 6579-1) were used as the agars to start the confirmation. The validated confirmation method can be used to

replace the biochemical confirmation under the following conditions:
— by laboratories using the ISO 6579-1; or

— by laboratories using an ISO 16140-2 validated alternative method that refers to ISO 6579-1 for confirmation; or

— by laboratories using an ISO 16140-2 validated alternative method that starts the confirmation from XLD

and/or BGA agar and/or the specified chromogenic agar.
The validated confirmation method cannot be used under the following conditions:

— by laboratories using an ISO 16140-2 validated alternative method that refers only to agars other than those

included in the validation to start the confirmation (e.g. Hektoen agar and SS agar only); or

— by laboratories using an ISO 16140-2 validated alternative method that refers only to a confirmation

procedure that does not require isolation on agar.

0.2 Validation and verification of methods for the microbiological confirmation and typing

procedures

The procedure described in this document is intended for the “full” validation of alternative

(proprietary) methods for microbiological confirmation and/or typing, hereafter referred to as

“alternative confirmation methods”.

During the validation study, the performance of the alternative confirmation method is compared to

the performance of the reference confirmation procedure.

The procedure for verification of alternative confirmation methods in a single laboratory is described

in ISO 16140-3.
© ISO 2019 – All rights reserved vii
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SIST EN ISO 16140-6:2020
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SIST EN ISO 16140-6:2020
INTERNATIONAL STANDARD ISO 16140-6:2019(E)
Microbiology of the food chain — Method validation —
Part 6:
Protocol for the validation of alternative (proprietary)
methods for microbiological confirmation and typing
procedures
1 Scope

This document specifies the general principle and the technical protocol for the validation of alternative

confirmation methods for microbiology in the food chain. This document compares the result of the

alternative confirmation method against the confirmation procedure of a reference method or, if

needed, a reference confirmation method (e.g. whole genome sequencing).

This document is applicable to the validation of alternative confirmation methods used for the analysis

(detection or quantification) of isolated microorganisms in:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production, handling;
— samples from the primary production stage.

Validated alternative confirmation methods can be used to replace (partly or completely) the

confirmation procedure described in:
— the reference method;

— an alternative method validated in accordance with ISO 16140-2 only if one of the isolation agars

specified in the validation study of the alternative confirmation method is used.

This document is also applicable to the validation of alternative typing methods, where the reference

method can be, for example, a serological method (e.g. serotyping of Salmonella) or a molecular method

(e.g. typing of Shiga toxin-producing E. coli).

This document is, in particular, applicable to bacteria and fungi. Some clauses can be applicable to other

(micro)organisms, to be determined on a case-by-case basis.

Validation studies in accordance with this document are primarily intended to be performed by

organizations or expert laboratories involved in method validation, but can also be used by a single

laboratory, especially when performing in-house validation under certain conditions (see ISO 16140-4).

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 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary

© ISO 2019 – All rights reserved 1
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SIST EN ISO 16140-6:2020
ISO 16140-6:2019(E)
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16140-1 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
acceptability limit

maximum positive or negative acceptable difference between the reference value (or if not known, the

accepted reference value) of a sample and an individual result obtained when applying the operating

procedure of an analytical method

Note 1 to entry: Annex D provides further information on the use of AL for this document.

[SOURCE: ISO 16140-1:2016, 2.1, modified — Note 1 to entry has been replaced.]
3.2
alternative confirmation or typing method
confirmation or typing method submitted for validation

method of analysis that confirms or types the same analyte as is confirmed or typed using the

corresponding reference method

Note 1 to entry: The method can be proprietary. The term “alternative” is used to refer to the entire “test

procedure and reaction system”. This term includes all ingredients, whether material or otherwise, required for

implementing the method.

Note 2 to entry: For clarity of reading, the text in this document generally describes validation of a confirmation

method (detailed examples are given in Annex B). If applicable, this can be read as validation of a typing method

(detailed examples are given in Annex C).
3.3
confirmation procedure

number of defined confirmation tests (3.4) that are performed on a strain, the combined results of which

are used to definitively confirm the identity of that strain
3.4
confirmation test
single test which is carried out to verify a presumptive result

Note 1 to entry: The result of a single test may not on its own be able to definitively confirm the identity of the strain.

[SOURCE: ISO 16140-1:2016, 2.17, modified — In the term and definition, “procedure or” has been

removed, “single” has been added and the Note 1 to entry has been replaced.]
3.5
microbial (sub)type

group of closely related microorganisms (within a species) distinguished by their shared specific

characteristics as determined by, for example, serological testing (serotype) or molecular testing

(genotype)
3.6
non-target strain

strain, defined according to the scope of the reference method, that would not reasonably be expected

to be confirmed by the alternative method
[SOURCE: ISO 16140-1:2016, 2.44, modified — In the definition, “confirmed” ha
...

SLOVENSKI STANDARD
oSIST prEN ISO 16140-6:2018
01-marec-2018
Mikrobiologija v prehranski verigi - Validacija metode - 6. del: Protokol za

validacijo alternativnih (lastniških) metod za postopke mikrobiološke potrditve in

tipizacije (ISO/DIS 16140-6:2017)

Microbiology of the food chain - Method validation - Part 6: Protocol for the validation of

alternative (proprietary) methods for microbiological confirmation and typing procedures

(ISO/DIS 16140-6:2017)

Mikrobiologie von Lebensmitteln und Futtermitteln - Verfahrensvalidierung - Teil 6:

Arbeitsvorschrift für die Validierung mikrobiologischer Nachweisverfahren (ISO/DIS

16140-6 )

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 6: Protocole

pour la validation des méthodes alternatives (propriétaires) pour confirmation et

identification microbiologique ( ISO/DIS 16140-6:2017)
Ta slovenski standard je istoveten z: prEN ISO 16140-6
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 16140-6:2018 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 16140-6:2018
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oSIST prEN ISO 16140-6:2018
DRAFT INTERNATIONAL STANDARD
ISO/DIS 16140-6
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2017-12-15 2018-03-09
Microbiology of the food chain — Method validation —
Part 6:
Protocol for the validation of alternative (proprietary)
methods for microbiological confirmation and typing
procedures
Microbiologie de la chaîne alimentaire — Validation des méthodes —

Partie 6: Protocole pour la validation des méthodes alternatives (propriétaires) pour confirmation et

identification microbiologique
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
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USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 16140-6:2017(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2017
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oSIST prEN ISO 16140-6:2018
ISO/DIS 16140-6:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

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ii © ISO 2017 – All rights reserved
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oSIST prEN ISO 16140-6:2018
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Contents

Foreword ......................................................................................................................................................................... 4

Introduction.................................................................................................................................................................... 5

1 Scope .............................................................................................................................................................................. 7

2 Normative references .............................................................................................................................................. 7

3 Terms and definitions ............................................................................................................................................. 8

4 General principles for the validation of confirmation and typing methods ........................................ 9

5 Strains ........................................................................................................................................................................ 10

6 Method comparison study .................................................................................................................................. 11

6.1 General ............................................................................................................................................................................ 11

6.2 Selection of test strains ............................................................................................................................................ 11

6.3 Inclusivity study ......................................................................................................................................................... 11

6.3.1 Testing of target strains ........................................................................................................................................ 11

6.3.2 Family level (non-Salmonella) ............................................................................................................................ 11

6.3.3 Genus level (non-Salmonella) ............................................................................................................................. 12

6.3.4 Species level (non-Salmonella) .......................................................................................................................... 12

6.3.5 Microbial (sub)type level (non-Salmonella) ................................................................................................. 12

6.3.6 Salmonella genus or species level ..................................................................................................................... 12

6.3.7 Salmonella serovar level ....................................................................................................................................... 12

6.4 Exclusivity study ......................................................................................................................................................... 13

6.4.1 Testing of non-target strains............................................................................................................................... 13

6.4.2 Family level (non-Salmonella) ............................................................................................................................ 13

6.4.3 Genus level (non-Salmonella) ............................................................................................................................. 13

6.4.4 Species level (non-Salmonella) .......................................................................................................................... 13

6.4.5 Microbial (sub)type level (non-Salmonella) ................................................................................................. 13

6.4.6 Salmonella genus and species level .................................................................................................................. 13

6.4.7 Salmonella serovar level ....................................................................................................................................... 14

6.5 Expression and interpretation of results ......................................................................................................... 14

6.6 Evaluation ..................................................................................................................................................................... 16

7 Interlaboratory study ........................................................................................................................................... 16

7.1 General ....................................................................................................................................................................... 16

7.2 Data sets to be obtained ..................................................................................................................................... 16

7.3 Protocol ..................................................................................................................................................................... 17

7.4 Expression of results ............................................................................................................................................ 18

7.5 Interpretation and evaluation .......................................................................................................................... 18

Annex A (normative) — Points to be considered when selecting strains for testing inclusivity

and exclusivity ............................................................................................................................................................. 20

A.1 General ................................................................................................................................................................................. 20

A.2 Target group categories ................................................................................................................................................ 20

A.3 Target group selection in inclusivity study ........................................................................................................... 20

A.4 Non-target groups selection in exclusivity study ............................................................................................... 20

Annex B (informative) — Example of an alternative confirmation method to the species level

(Listeria monocytogenes) ......................................................................................................................................... 22

Annex C (informative) — Example of an alternative typing method to the Salmonella serovar

level (15 different serovars claimed) .................................................................................................................. 25

Annex D (normative) — Overview of the various options for validation under this part of

ISO 16140 and the acceptability limits ............................................................................................................... 28

Bibliography ................................................................................................................................................................ 30

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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 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 the following

URL: www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,

Microbiology (Working Group WG 3, Method validation).
A list of all parts of the ISO 16140 series can be found on the ISO website.
4 © ISO 2017 – All rights reserved
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Introduction

The ISO 16140 series has been elaborated in response to the need for various ways to validate or verify

test methods. It is the successor of ISO 16140:2003, Microbiology of food and animal feeding stuffs —

Protocol for the validation of alternative methods. ISO 16140 series consists of several parts with the

general title, Microbiology of the food chain — Method validation:
 Part 1: Vocabulary

 Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method

 Part 3: Protocol for the verification of reference and validated alternative methods implemented in a

single laboratory
 Part 4: Protocol for single-laboratory (in-house) method validation

 Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods

 Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological

confirmation and typing procedures

ISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment or

[7]

revision of a standardized reference method , is a closely linked International Standard. This

International Standard, which establishes technical rules for the development and validation of

standardized methods, is intended for the development of standardized methods by ISO/TC 34, Food

products, Subcommittee SC 9, Microbiology and CEN/TC 275/WG 6, Microbiology of the food chain.

In general two stages are needed before a method can be used in a laboratory:

 The first stage is the validation of the method. This is either conducted in several laboratories

(parts 2 and 5 of ISO 16140) or in one laboratory (part 4 of ISO 16140).

 The second stage is method verification, where a laboratory demonstrates that it can satisfactorily

perform a validated method. This is described in part 3 of ISO 16140 (method verification). In

part 3, a separation is made between verification of (food) items that are included in the validation

study and (food) items that are not tested in the validation study but belong within the scope of

validation.

NOTE 1 Standardized reference methods (with and without published validation data) only require verification

before implementation in the laboratory.

NOTE 2 In this part of ISO 16140, the word ‘item’ is sometimes combined with ‘food’ to improve the readability

of this document. However, the word ‘food’ is interchangeable with ‘feed’ and the other areas of the food chain as

mentioned in the Scope of ISO 16140-6.

Part 4 of ISO 16140 addresses validation within a single laboratory. The results are therefore only valid

in the laboratory which conducted the study. In this case, verification (part 3 of ISO 16140) is not

required.

Part 5 of ISO 16140 describes protocols for situations where a more rapid validation is required or

when the method to be validated is highly specialised, and, the number of participating laboratories

required by ISO 16140-2 cannot be reached.
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The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above.

It also guides the users in selecting the right part of the ISO 16140 series, taking into account the

purpose of the study and the remarks given above. For this, it is important to distinguish between

'reference method' and 'standardized reference method'. A reference method is an internationally

recognized and widely accepted method (term 2.59 of ISO 16140-1:2016) and a standardized reference

method is a reference method described in a standard (term 3.5 of ISO 17468:2016). In the ISO 16140

series, reference method includes standardized reference method. The flow diagram acknowledges that

published validation data may not be available for some standardized reference methods.

Figure 1 — Flow chart for application of the different parts of the ISO 16140-series

Part 6 of ISO 16140, is somewhat different from the other parts in the ISO 16140 series in that it relates

to a very specific situation where only the confirmation procedure of a method is validated. The

confirmation procedure advances a suspected (presumptive) result to a confirmed positive result. The

typing of pure strains (e.g. serotyping of Salmonella) is included in part 6 of ISO 16140.

The procedure described in this part of ISO 16140 is intended for the ‘full’ validation of alternative

(proprietary) methods for microbiological confirmation and typing, further referred to as ‘alternative

confirmation methods’. The procedure for verification of alternative confirmation methods in a single

laboratory will be described in part 3 of ISO 16140.

During the validation study, the performance of the alternative confirmation method will be compared

to the performance of the reference confirmation procedure.
6 © ISO 2017 – All rights reserved
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Microbiology of the food chain — Method validation — Part 6:
Protocol for the validation of alternative (proprietary) methods
for microbiological confirmation and typing procedures
1 Scope

This part of ISO 16140 specifies the general principle and the technical protocol for the validation of

alternative, mostly proprietary, confirmation methods for microbiology in the food chain. This part of

ISO 16140 compares the result of the alternative confirmation method against that of the reference

confirmation procedure.

This part of ISO 16140 is applicable to the validation of alternative confirmation methods used in the

analysis (detection or quantification) of microorganisms in
 products intended for human consumption,
 products intended for animal feeding,
 environmental samples in the area of food and feed production, handling, and
 samples from the primary production stage.

Validated alternative confirmation methods can be used to replace (partly or completely) the

confirmation procedure described in:
 the reference method; and
[5]

 an alternative method validated according to part 2 of ISO 16140 as long as one of the isolation

agars specified in the validation study of the alternative confirmation method is used.

This part of ISO 16140 is also applicable to the validation of alternative typing methods, where the

reference method for example can be a serological method (e.g. serotyping of Salmonella) or a

molecular method (e.g. typing of Shiga toxin-producing E. coli).

This part of ISO 16140 is in particular applicable to bacteria and fungi. Some clauses can be applicable

to other (micro)organisms, to be determined on a case-by-case-basis.

Validation studies according to this part of ISO 16140 are intended to be performed by organizations

involved in method validation and not by single user laboratories.
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 food and animal feeding stuffs — General requirements and guidance for

microbiological examinations

ISO 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary

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3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16140-1 and the following

apply. ISO and IEC maintain terminological databases for use in standardization at the following

addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
acceptability limit

maximum positive or negative acceptable difference between the reference value (or if not known, the

accepted reference value) of a sample and an individual result obtained when applying the operating

procedure of an analytical method
[SOURCE: ISO 16140-1:2016, 2.1, modified]

Note 1 to entry: Annex D provides further information on the use of acceptability limit (AL) for this part of

ISO 16140.
3.2
confirmation procedure

number of defined tests that are performed on a strain, the combined results of which are used to

definitively confirm the identity of that strain
3.3
confirmation test
single test which is carried out to verify a presumptive result
[SOURCE: ISO 16140-1:2016, 2.17, modified]

Note 1 to entry: The results of a single test may not on its own be able to definitively confirm the identity of the

strain.
3.4
non-target strain

strain, defined according to the scope of the reference method that would not reasonably be expected to

be confirmed by the alternative method
[SOURCE: ISO 16140-1:2016, 2.44, modified]
3.5
reference confirmation or typing procedure

combination of the confirmation or typing tests that are claimed to be replaced by the alternative

confirmation or typing method

Note 1 to entry: The number of confirmation tests depends on the reference method for the specific

microorganisms. The number of confirmation tests can also be one.
Note 2 to entry: A detailed example is given in Annex B and in Annex C.

Note 3 to entry: For clarity of reading the text of this document is generally described for validation of a

confirmation procedure (Annex B). If applicable this can be read as typing procedure (Annex C).

8 © ISO 2017 – All rights reserved
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3.6
microbial (sub)type

a group of closely related microorganisms (within a species) distinguished by their shared specific

characteristics as determined by e.g. serological testing (serotype) or molecular testing (genotype)

3.7
target strain

strain, defined according to the scope of the reference method, that is expected to be confirmed by the

alternative method
[SOURCE: ISO 16140-1:2016, 2.74, modified]
3.8
typing
the process of determining a particular microbial (sub)type
4 General principles for the validation of confirmation and typing methods

In the validation study, the alternative confirmation method is compared to the confirmation procedure

described in the reference method for the enumeration or detection of specific (groups of)

microorganisms.
The validation protocol comprises two phases:

 a method comparison study of the alternative confirmation method against the reference

confirmation procedure carried out in the organizing laboratory (Clause 6);
 an interlaboratory study (Clause 7).

The validation protocol shall clearly define the selective agars from which strains can be confirmed

using the alternative confirmation method. If successfully validated, the alternative confirmation

method can only be used if strains are recovered on an agar that was used and shown to be acceptable

within the validation study. Figure 2 shows the possibilities where an alternative confirmation method

validated according to part 6 of ISO 16140 can be applied (see text in red).

Figure 2 — Use of alternative confirmation methods validated according to part 6 of ISO 16140

(see text in red)
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EXAMPLE Application of a validated alternative confirmation method:

An alternative confirmation method based on PCR is validated to replace the biochemical confirmation for

[1]

Salmonella as described in ISO 6579-1 . In the validation study XLD (mandatory agar according to ISO 6579-1)

and BGA (optional agar according to ISO 6579-1) were used as the agars to start the confirmation. The validated

confirmation method can be used under the following conditions:

1) by laboratories using the ISO 6579-1 to replace the biochemical confirmation, or

2) by laboratories using an ISO 16140-2 validated alternative method that refers to ISO 6579-1 for (biochemical)

confirmation, or

3) by laboratories using an ISO 16140-2 validated alternative method that starts the confirmation from XLD

and/or BGA agar.
The validated confirmation method cannot be used under the following conditions:

1) by laboratories using an ISO 16140-2 validated alternative method that refers to a proprietary method for

confirmation (e.g. a chromogenic agar), or

2) by laboratories using an ISO 16140-2 validated alternative method that refers to other agars to start the

confirmation (e.g. Hektoen agar), or

3) by laboratories using an ISO 16140-2 validated alternative method that refers to a confirmation procedure

not based on isolation on agar (e.g. an ELISA test).
[6]

NOTE It is possible to include data already obtained in an inclusivity or exclusivity part of an ISO 16140-2

study into an ISO 16140-6 study, if relevant.

The technical rules for performing the method comparison study and the interlaboratory study are

given in Clause 6 and Clause 7. The following 6 cases will be covered; a distinction is made between the

confirmation/typing of Salmonella and that of other microorganisms.

 validation of methods used for confirmation to the Family level (non-Salmonella);

 validation of methods used for confirmation to the Genus level (non-Salmonella);

 validation of methods used for confirmation to the Species level (non-Salmonella);

 validation of methods used for confirmation/typing to the Microbial (sub)type level (non-

Salmonella);

 validation of methods used for confirmation/typing to the Salmonella genus or species level;

 validation of methods used for confirmation/typing to the Salmonella serovar level.

5 Strains

The pure strains used for determining the inclusivity and the exclusivity have to be well characterised:

therefore, strain characterisation shall follow the requirements as specified in ISO 7218. This

identification information of each strain will be used to (additionally) confirm the result in cases of

discrepancies between the results of the reference confirmation procedure and the alternative

confirmation method.
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6 Method comparison study
6.1 General

The method comparison study is the part of the validation that is performed in one laboratory. It

consists of an inclusivity and exclusivity study of the alternative confirmation method. The results are

then compared to those of the reference confirmation procedure.
6.2 Selection of test strains

A range of strains shall be used. Criteria for selecting test strains are given in Annex A. The strains

selected should take into account the measurement principle of the alternative method (e.g. culture-

based, immunological, molecular-based). Different measurement principles may require the use of

panels of different test strains. It is important to include suspect and interfering colonies that may grow

on the media used for the reference method.

The rationale on the choice of the strains and their characteristics has to be included in the study

report.

Each strain should be characterised biochemically and/or serologically and/or genetically in sufficient

detail for its identity to be known. Strains should preferably have been isolated from foods, feed, the

food-processing environment, or from primary production: depending on the scope of the validation.

However, clinical, environmental and culture collection strains can also be used. The original source of

all strains should be known and they shall be held in a local (e.g. expert laboratory), national or

[3]

international culture collection to enable them to be used in future testing if required. See ISO 11133

for local maintenance of stock cultures.

Reference method results generated by a specialised reference laboratory can be used if the laboratory

performing the validation study is not able to perform the confirmati
...

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