National foreword

This British Standard is the UK implementation of EN ISO 16140‑7:2024. It is identical to ISO 16140‑7:2024.

The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.

A list of organizations represented on this committee can be obtained on request to its committee manager.

Contractual and legal considerations

This publication has been prepared in good faith, however no representation, warranty, assurance or undertaking (express or implied) is or will be made, and no responsibility or liability is or will be accepted by BSI in relation to the adequacy, accuracy, completeness or reasonableness of this publication. All and any such responsibility and liability is expressly disclaimed to the full extent permitted by the law.

This publication is provided as is, and is to be used at the recipient’s own risk.

The recipient is advised to consider seeking professional guidance with respect to its use of this publication.

This publication is not intended to constitute a contract. Users are responsible for its correct application.

Compliance with a British Standard cannot confer immunity from legal obligations.

This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 November 2024.

This European Standard was approved by CEN on 6 October 2024.

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, Czechia, 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 foreword

This document (EN ISO 16140‑7:2024) 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 May 2025, and conflicting national standards shall be withdrawn at the latest by May 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 16140‑7:2024 has been approved by CEN as EN ISO 16140‑7:2024 without any modification.

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 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.

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 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 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;
Part 7: Protocol for the validation of identification methods of microorganisms.

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, ISO 16140‑6 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 17468:2023, 3.7, 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 (regarding qualitative and quantitative methods) and ISO 16140‑5 (regarding quantitative methods only) can also be used for validation without a reference method.

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.

NOTE 1 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.

ISO 16140‑6 and this document (ISO 16140‑7) are somewhat different from the other parts in the ISO 16140 series in that they relate to very specific situations.

ISO 16140‑6 is restricted to the confirmation procedure of a method 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 ISO 16140‑6. The validation study in ISO 16140‑6 clearly specifies 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 was shown to be acceptable within the validation study. Figure 2 shows the possibilities where an alternative confirmation method validated in accordance with ISO 16140‑6 can be applied (see text in the boxes).

EXAMPLE 1 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 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.

This document (ISO 16140‑7) addresses the validation of identification procedures (e.g. molecular identification using multiplex PCR or DNA sequencing or mass spectrometry). This document differs from the other parts in the ISO 16140 series, as it is intended for microbial identification for which there is no reference method and, therefore, it is not possible to run a method comparison study. The validation study in this document specifies the identification method principle, the identification database and algorithm when appropriate, and the agar(s) from which strains can be identified. If properly characterized and successfully validated, the identification method can only be validly used on strains recovered on the agars covered and shown to have been acceptable within the validation study.

NOTE 2 Whole-genome sequencing (WGS) in accordance with ISO 23418 will eventually be a reference method for all microorganisms, but the implementation of this technique is still at an early stage. Therefore, the use of WGS cannot currently be requested as a reference method for a large panel of strains.

Figure 3 shows the possibilities where an alternative confirmation method validated in accordance with ISO 16140‑6 and an alternative identification method validated in accordance with this document can be applied within a reference method or an ISO 16140‑2 validated detection or enumeration method. The result provided by the ISO 16140‑7 validated method can be considered as additional information on the identity of the tested colony(ies); this result cannot be taken as a confirmation result. When there is a discrepancy between the results of the ISO 16140‑6 validated method and the ISO 16140‑7 validated method, a root cause analysis is conducted. An ISO 16140‑7 validated method can also be used to identify colonies within methods that do not require a confirmation step.

If the identification method is also validated in accordance with ISO 16140‑6, the same method can be used for both confirmation and identification.

When a confirmation method is used, it is possible to apply an identification method validated in accordance with this document for further identification.

EXAMPLE 2 An alternative confirmation method of Campylobacter genus can be validated in accordance with ISO 16140‑6 and compared to the mandatory confirmation procedure at the genus level described in ISO 10272‑1. The identification at the Campylobacter species level is optional in ISO 10272‑1 and ISO 10272‑2 and is therefore not mandatory. In this instance, an identification method at the Campylobacter species level can be validated in accordance with this document. If the method is validated by ISO 16140‑6 and this document, it can be used for both confirmation and identification purposes.

0.2 Validation and verification of identification methods of microorganisms

The procedure described in this document is intended for validation of identification methods of microorganisms. This procedure comprises two parts: a performance characteristics study and an interlaboratory study.

The procedure for validation of identification methods of microorganisms in a single laboratory is described in ISO 16140‑4. The procedure for verification of identification methods of microorganisms in a single laboratory is described in ISO 16140‑3.

1 Scope

This document specifies the general principle and the technical protocol for the validation of identification methods of microorganisms for microbiology in the food chain. As there is no reference method, no method comparison study can be run. Therefore, this document provides a protocol to evaluate the performance characteristics and validate the method workflow using well-defined strains. When required, an additional identification method can be used.

This document is applicable to the validation of identification methods of microorganisms that are used for the analysis of isolated colonies from:

products intended for human consumption;
products for feeding animals;
environmental samples in the area of food and feed production and handling;
samples from the primary production stage.

Identification methods only validated in accordance with this document cannot be used instead of confirmation described in:

the reference method;
an alternative method validated in accordance with ISO 16140‑2;
an alternative method validated in accordance with ISO 16140‑6.

In these instances, the identification method is validated in accordance with ISO 16140‑6 method that is used as a confirmation method.

This document is applicable to bacteria and fungi. Some clauses can be applicable to other (micro)organisms, which can be determined on a case-by-case basis.

2 Normative references

3 Terms and definitions

4 General principles for the validation of identification methods of microorganisms

5 Strains

6 Performance characteristics of an identification method

6.1 General

6.2 Description of the concept and limitation(s) of the identification method

6.3 Identification reliability of the identification method

6.4 Evaluation

7 Interlaboratory study

7.1 General

7.2 Data sets to be obtained

7.3 Protocol

7.4 Expression of results

7.5 Interpretation and evaluation

Bibliography

BS EN ISO 16140-7:2024

Microbiology of the food chain. Method validation - Protocol for the validation of identification methods of microorganisms

Current

Published:

30 Nov 2024