ISO/TS 16099:2025

Water quality — Polymerase chain reaction (PCR) for the detection and quantification of microorganisms and viruses — General requirements, quality assurance and validation

This document specifies the general requirements for the in vitro amplification of nucleic acid sequences (DNA or RNA). This includes polymerase chain reaction (PCR)-based methods like quantitative PCR, qualitative PCR, reverse transcription-PCR and digital PCR. The minimum requirements laid down in this document are intended to ensure that comparable and reproducible results are obtained in different organizations. It covers quality assurance aspects to be considered when working with PCR-based methods in a laboratory as well as validation and verification. In addition to laboratory PCR-based methods, this document is also applicable to on-site PCR-based methods. This document is applicable to PCR-based methods used for the analysis of microorganisms and viruses in different water matrices, including but not limited to: — drinking water; — groundwater; — pool water; — process water; — surface water; — wastewater. This document is applicable to the detection and quantification of nucleic acids (DNA or RNA) of microorganisms by PCR-based methods in water such as bacteria, yeasts, fungi but also parasites such as Cryptosporidium, Giardia, amoebas and multicellular organisms. In addition, this document is applicable to the detection and quantification of nucleic acids from viruses in water by PCR-based methods. NOTE In the context of this document, viruses are considered to be microorganisms. Clauses in this document can also specifically apply to viruses and not to other types of microorganisms. In these clauses, viruses are mentioned separately.

Qualité de l'eau — Réaction de polymérisation en chaîne (PCR) pour la détection et la quantification des microorganismes et des virus — Exigences générales, assurance de la qualité et validation

General Information

• Status: Published
• Publication Date: 22-Jul-2025
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: ISO/TC 147/SC 4 - Microbiological methods
• Drafting Committee: ISO/TC 147/SC 4 - Microbiological methods
• Current Stage: 6060 - International Standard published
• Start Date: 23-Jul-2025
• Due Date: 22-Apr-2025
• Completion Date: 23-Jul-2025

Standards Content (Sample)

Technical
Specification
ISO/TS 16099
First edition
Water quality — Polymerase chain
2025-07
reaction (PCR) for the detection and
quantification of microorganisms
and viruses — General
requirements, quality assurance
and validation
Qualité de l'eau — Réaction de polymérisation en chaîne (PCR)
pour la détection et la quantification des microorganismes et des
virus — Exigences générales, assurance de la qualité et validation
Reference number
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
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: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .vi
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle .11
4.1 General .11
4.2 Test material .11
4.3 Sampling, transport and storage. 12
4.4 Preparation of the sample . 12
4.4.1 General . 12
4.4.2 Preparation and concentration of samples . 12
4.4.3 Treatment of samples containing heavy metal particles . 13
5 Nucleic acid extraction . 14
5.1 General .14
5.2 Nucleic acid removal or inactivation from damaged microorganism(s).14
5.3 Nucleic acid quantity and quality . 15
5.4 Stability of nucleic acid extracts . 15
6 PCR-based methods . 16
6.1 General .16
6.2 Qualitative PCR-based methods .17
6.3 Quantitative PCR-based methods .17
6.4 Digital PCR.17
7 Laboratory setup .18
7.1 General .18
7.2 Layout of laboratory areas and workflow .18
7.3 Working environment .19
7.3.1 General .19
7.3.2 Reagents preparation area . 20
7.3.3 Sample preparation area . 20
7.3.4 PCR area .21
7.4 Cleaning of laboratory .21
7.5 Environmental monitoring for PCR .21
8 Equipment .21
8.1 General .21
8.2 Biological safety cabinet . 22
8.3 Centrifuge . 22
8.4 Digital PCR system . 22
8.5 Filtration setup. 23
8.6 Freezer and ultra-low temperature freezer . 23
8.7 Heating block module . 23
8.8 PCR workstation . 23
8.9 Pipettes .24
8.10 Pipetting robots (optional) .24
8.11 Refrigerators .24
8.12 Thermal cycler .24
8.13 Spectrophotometry or fluorometry instrument . 25
8.14 On-site PCR systems . 25
9 Reagents and consumables .25
9.1 General . 25
9.2 Primers and probes . 26

iii
9.2.1 General . 26
9.2.2 Quality control . 26
9.2.3 Storage . 26
9.3 Lyophilized PCR reagents . 26
9.4 Ammonium oxalate solution .27
9.5 Pipetting tips .27
9.6 Membrane filters .27
9.7 PCR plates and tubes .27
9.8 Calibration standard .27
9.9 Master mix . 28
9.9.1 General . 28
9.9.2 Commercially available master mixes . 28
9.9.3 Master mix prepared by user . 28
9.10 Chemicals and consumables for nucleic acid extraction kits . 29
9.10.1 General .
...

FINAL DRAFT
Technical
Specification
ISO/DTS 16099
ISO/TC 147/SC 4
Water quality — Polymerase chain
Secretariat: DIN
reaction (PCR) for the detection and
Voting begins on:
quantification of microorganisms
2025-03-27
and viruses — General
Voting terminates on:
requirements, quality assurance
2025-05-22
and validation
Qualité de l'eau — Réaction de polymérisation en chaîne (PCR)
pour la détection et la quantification des microorganismes et des
virus — Exigences générales, assurance de la qualité et validation
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 SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
ISO/DTS 16099:2025(en) © ISO 2025

FINAL DRAFT
ISO/DTS 16099:2025(en)
Technical
Specification
ISO/DTS 16099
ISO/TC 147/SC 4
Water quality — Polymerase chain
Secretariat: DIN
reaction (PCR) for the detection and
Voting begins on:
quantification of microorganisms
and viruses — General
Voting terminates on:
requirements, quality assurance
and validation
Qualité de l'eau — Réaction de polymérisation en chaîne (PCR)
pour la détection et la quantification des microorganismes et des
virus — Exigences générales, assurance de la qualité et validation
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 SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ISO/DTS 16099:2025(en) © ISO 2025

ii
ISO/DTS 16099:2025(en)
Contents Page
Foreword .vi
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle .11
4.1 General .11
4.2 Test material .11
4.3 Sampling, transport and storage. 12
4.4 Preparation of the sample . 12
4.4.1 General . 12
4.4.2 Preparation and concentration of samples . 12
4.4.3 Treatment of samples containing heavy metal particles . 13
5 Nucleic acid extraction . 14
5.1 General .14
5.2 Nucleic acid removal or inactivation from damaged microorganism(s).14
5.3 Nucleic acid quantity and quality . 15
5.4 Stability of nucleic acid extracts . 15
6 PCR-based methods . 16
6.1 General .16
6.2 Qualitative PCR-based methods .17
6.3 Quantitative PCR-based methods .17
6.4 Digital PCR.17
7 Laboratory setup .18
7.1 General .18
7.2 Layout of laboratory areas and workflow .18
7.3 Working environment .19
7.3.1 General .19
7.3.2 Reagents preparation area . 20
7.3.3 Sample preparation area . 20
7.3.4 PCR area .21
7.4 Cleaning of laboratory .21
7.5 Environmental monitoring for PCR .21
8 Equipment .21
8.1 General .21
8.2 Biological safety cabinet . 22
8.3 Centrifuge . 22
8.4 Digital PCR system . 22
8.5 Filtration setup. 23
8.6 Freezer and ultra-low temperature freezer . 23
8.7 Heating block module . 23
8.8 PCR workstation . 23
8.9 Pipettes .24
8.10 Pipetting robots (optional) .24
8.11 Refrigerators .24
8.12 Thermal cycler .24
8.13 Spectrophotometry or fluorometry instrument . 25
8.14 On-site PCR systems . 25
9 Reagents and consumables .25
9.1 General . 25
9.2 Primers and probes . 26

iii
ISO/DTS 16099:2025(en)
9.2.1 General . 26
9.2.2 Quality control . 26
9.2.3 Storage . 26
9.3 Lyophilized PCR reagents . 26
9.4 Ammonium oxalate solution .27
9.5 Pipetting tips .27
9.6 Membrane filters .
...

ISO/DTS 16099
ISO/DTS 16099
ISO/TC 147/SC 4
Secretariat: DIN
Date: 20242025-03-12-04
Water quality — Polymerase chain reaction (PCR) for the
detection and quantification of microorganisms and viruses —
General requirements, quality assurance and validation

Draft DTS
Warning for WDs, CDs and draft DTSs
This document is not an ISO Technical Specification. It is distributed for review and comment. It is subject to
change without notice and may not be referred to as an ISO Technical Specification.
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/DTS 16099
Qualité de l'eau — Réaction de polymérisation en chaîne (PCR) pour la détection et la quantification des
microorganismes et des virus — Exigences générales, assurance de la qualité et validation
2 © ISO #### – All rights reserved

ISO/DTS 16099:(en)
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
EmailE-mail: [e-mail]
Website: www.iso.orgwww.iso.org
Published in Switzerland
iii
ISO/DTS 16099:(en)
Contents
Foreword . vi
Introduction . vii
1 Scope . 2
2 Normative references . 2
3 Terms and definitions . 3
4 Principle . 14
4.1 General. 14
4.2 Test material . 14
4.3 Sampling, transport and storage . 15
4.4 Preparation of the sample . 15
5 Nucleic acid extraction . 17
5.1 General. 17
5.2 Nucleic acid removal or inactivation from damaged microorganism(s) . 18
5.3 Nucleic acid quantity and quality . 18
5.4 Stability of nucleic acid extracts . 18
6 PCR-based methods . 19
6.1 General. 19
6.2 Qualitative PCR-based methods . 20
6.3 Quantitative PCR-based methods . 20
6.4 Digital PCR . 21
7 Laboratory setup . 21
7.1 General. 21
7.2 Layout of laboratory areas and workflow . 22
7.3 Working environment . 23
7.4 Cleaning of laboratory . 25
7.5 Environmental monitoring for PCR . 26
8 Equipment . 26
8.1 General. 26
8.2 Biological safety cabinet . 26
8.3 Centrifuge . 26
8.4 Digital PCR system . 27
8.5 Filtration setup . 27
8.6 Freezer and ultra-low temperature freezer . 27
8.7 Heating block module . 28
8.8 PCR workstation . 28
8.9 Pipettes . 28
8.10 Pipetting robots (optional) . 29
8.11 Refrigerators . 29
8.12 Thermal cycler . 29
8.13 Spectrophotometry or fluorometry instrument . 29
8.14 On-site PCR systems . 30
9 Reagents and consumables . 30
9.1 General. 30
9.2 Primers and probes. 30
9.3 Lyophilized PCR reagents . 31
9.4 Ammonium oxalate solution . 31
9.5 Pipetting tips . 31
iv
ISO/DTS 16099:(en)
9.6 Membrane filters. 32
9.7 PCR plates and tubes . 32
9.8 Calibration standard . 32
9.9 Master mix . 32
9.10 Chemicals and consumables for nucleic acid extraction kits . 34
9.11 On-site PCR . 34
10 Procedure . 35
10.1 Controls . 35
10.2 Data analysis of results . 38
10.3 Evaluation of results . 39
10.4 Test report . 42
11 Validation and verification of PCR-based methods . 43
11.1 General. 43
11.2 Pre-validation . 44
11.3 Validation . 45
11.4 Sample preparation . 46
11.5 Water matrices . 47
11.6 Performance characteristics for validation . 48
11.7 Validation of the PCR step . 49
11.8 Validation of qualitative PCR-based methods . 52
11.9 Validation of quantitative PCR-based methods . 53
11.10 Controls and validation . 56
11.11 Interlaboratory study . 56
11.12 Verification of PCR-based methods . 57
Annex A (informative) Example of an interpretation of qualitative PCR results for Escherichia
coli . 58
Annex B (informative) Example of an interpretation of quantitative PCR results for Legionella
pneumophila with an internal control . 60
Annex C (informative) Example of an interpretation of dPCR results for SARS-CoV-2 . 67
Annex D (informative) Verification of the calibration function of the quantitative PCR phase . 71
Bibliography . 80

v
ISO/DTS 16099:(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrot
...