ISO/TS 16099:2025

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
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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 . 29
9.10.2 Commercially available extraction kits . 29
9.10.3 Nucleic acid extraction chemicals prepared by user . 29
9.11 On-site PCR . 29
10 Procedure .30
10.1 Controls . 30
10.1.1 General . 30
10.1.2 Negative process control . 30
10.1.3 Positive process control .31
10.1.4 Internal process control .31
10.1.5 Amplification control .31
10.1.6 Positive PCR control .32
10.1.7 Negative PCR control .32
10.1.8 Required controls for dPCR .32
10.2 Data analysis of results . 33
10.2.1 Data analysis for real-time PCR . 33
10.2.2 Data analysis for dPCR . 33
10.3 Evaluation of results . . 34
10.3.1 General . 34
10.3.2 Evaluation of positive controls using control charts . 35
10.3.3 Standard curve evaluation . 35
10.3.4 Absolute quantification (real-time PCR and dPCR) . 36
10.3.5 Relative quantification . 36
10.4 Test report .37
11 Validation and verification of PCR-based methods.37
11.1 General .37
11.2 Pre-validation . 38
11.3 Validation . 39
11.3.1 General . 39
11.3.2 Method comparison studies . 39
11.3.3 Validation without method comparison. 40
11.4 Sample preparation . 40
11.5 Water matrices .41
11.6 Performance characteristics for validation .41
11.7 Validation of the PCR step .43
11.7.1 General .43
11.7.2 Multiplex PCR-related methods .43
11.7.3 Calibration of standard curve.43
11.7.4 Measurement range . 44
11.7.5 Inclusivity and exclusivity . 44
11.8 Validation of qualitative PCR-based methods.45
11.8.1 General .45
11.8.2 Sensitivity .45
11.8.3 (Relative) trueness .45

iv
11.8.4 (Relative) limit of detection . 46
11.9 Validation of quantitative PCR-based methods . 46
11.9.1 General . 46
11.9.2 (Relative) trueness . 46
11.9.3 (Relative) limit of quantification .47
11.9.4 (Relative) limit of detection .47
11.9.5 Linearity .47
11.9.6 Specificity and sensitivity . 48
11.9.7 Precision . 48
11.9.8 Robustness . . 48
11.10 Controls and validation . 49
11.11 Interlaboratory study . 49
11.12 Verification of PCR-based methods . 49
Annex A (informative) Example of an interpretation of qualitative PCR results for Escherichia
coli . .51
Annex B (informative) Example of an interpretation of quantitative PCR results for Legionella
pneumophila with an internal control .53
Annex C (informative) Example of an interpretation of dPCR results for SARS-CoV-2 .57
Annex D (informative) Verification of the calibration function of the quantitative PCR phase .60
Bibliography .68

v
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 4,
Microbiological methods.
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.

vi
Introduction
PCR-based methods are developed for the detection and/or quantification of, for example, pathogenic
bacteria, for rapid and reliable outcomes as an alternative to culture-based methods. For example, for the
screening on the presence of Legionella or faecal-related microorganisms in water, see References [26], [29],
[43], [49] and [56] for further information.
Performing nucleic acid quantification assays to a high standard of analytical quality can be challenging.
For example, it is well known that impure or degraded nucleic acid extracts can affect the accuracy of
quantification. Similarly, a poorly designed quantitative polymerase chain reaction (qPCR) assay with poor
amplification efficiency and poor primer specificity will impact the quantification accuracy of nucleic acid
targets.
In addition, aspects such as the water matrix and standard curves can have a significant influence on the
accuracy of quantitative measurements of nucleic acid targets. Therefore, it is important to improve the
reliability of data by setting general requirements for PCR-based methods.

vii
Technical Specification ISO/TS 16099:2025(en)
Water quality — Polymerase chain reaction (PCR) for the
detection and quantification of microorganisms and viruses
— General requirements, quality assurance and validation
1 Scope
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.
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 19458, Water quality — Sampling for microbiological analysis
ISO 20836, Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of microorganisms
— Thermal performance testing of thermal cyclers
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
5′-3′-exonuclease activity
ability of deoxyribonucleic acid (DNA) polymerase (3.18) to cleave nucleotides in the 5′-3′-direction
Note 1 to entry: When a fluorescent probe (3.50) is used for the detection of amplification, the 5’-3’-exonuclease
activity of DNA polymerase allows the probe to hydrolyse and emit fluorescence.
3.2
absolute quantification by digital polymerase chain reaction
absolute quantification by dPCR
procedure involving PCR amplification and target copy quantification which does not require a standard
curve to determine the concentration of a target nucleic acid (3.31) in a sample
[SOURCE: ISO 22174:2024, 3.7.9]
3.3
absolute quantification by real-time polymerase chain reaction
absolute quantification by real-time PCR
procedure involving PCR amplification to determine the concentration of a target nucleic acid (3.31) in a
sample by comparison with a standard curve, derived from standards containing a defined amount of target
[SOURCE: ISO 22174:2024, 3.6.4]
3.4
aliquot
portion of a quantity of liquids which has been divided into separate parts at the same time under identical
conditions
3.5
amplification control
nucleic acid (3.31) added in a defined amount or copy number which serves as a control for amplification
Note 1 to entry: This nucleic acid sequence (3.34) can be endogenous [naturally present in the tested matrix (3.25)] or
exogenous (naturally absent in the tested matrix).
Note 2 to entry: The amplification control can either be an internal or external control. With the internal amplification
control, the control is added to each polymerase chain reaction (PCR); this requires the use of a multiplex PCR (3.26).
The external amplification control is added to each aliquot of the extracted nucleic acid serving as a control for
amplification in a separate reaction.
Note 3 to entry: An exogenous internal amplification control can be homologous (amplified using the same primers
(3.47) as used for amplification of the target) or heterologous (amplified using different primers than those used for
amplification of the target). A homologous internal amplification control amplicon shall be distinguishable from the
microbial target amplicon (e.g. by size or by insertion of a different probe-binding sequence).
3.6
annealing
pairing of complementary single strands of nucleic acids (3.31) to form a double-stranded molecule
Note 1 to entry: Lowering the temperature of the PCR reaction allows primers (3.47) and probes (3.50) to pair with a
complementary single-stranded nucleic acid to form a double-stranded molecule.
[SOURCE: ISO 22174:2024, 3.4.13, modified — Note 1 to entry has been added.]

3.7
background fluorescence
background
intrinsic level of fluorescence resulting from the reagents, consumables and instruments used
[SOURCE: ISO 22174:2024, 3.4.7]
3.8
complementary DNA
cDNA
single-stranded deoxyribonucleic acid (DNA) (3.12), complementary to a given ribonucleic acid (RNA) (3.63)
and synthesised in the presence of reverse transcriptase to serve as a template for DNA amplification (3.17)
[SOURCE: ISO 20395:2019, 3.5]
3.9
cross-contamination
unintended transfer of nucleic acids (3.31) [deoxyribonucleic acid (DNA) (3.12) or ribonucleic acid (RNA) (3.63)]
EXAMPLE Cross-contamination can occur between samples during PCR preparation.
3.10
decontamination
procedure to remove or reduce nucleic acids (3.31) and/or nucleases from materials and surfaces
3.11
denaturation
process which results in the separation of the double-stranded nucleic acid (3.31) into single-stranded
nucleic acids
[SOURCE: ISO 22174:2024, 3.4.11]
3.12
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA) form
[SOURCE: ISO 22174:2024, 3.1.6]
3.13
deoxyribonuclease
DNase
enzyme which degrades deoxyribonucleic acid (DNA) (3.12)
[SOURCE: ISO 22174:2024, 3.1.7]
3.14
deoxyribonucleoside triphosphate
dNTP
solution containing deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),
deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP) and/or deoxyuridine
triphosphate (dUTP)
[SOURCE: ISO 22174:2024, 3.4.2]
3.15
detection of polymerase chain reaction product
detection of PCR product
detection of amplicon
process which signals the presence of a PCR product
[SOURCE: ISO 22174:2024, 3.1.15, modified — “an amplicon” has been replaced by “a PCR product”.]

3.16
digital polymerase chain reaction
digital PCR
dPCR
procedure in which nucleic acid (3.31) templates are randomly and independently distributed across multiple
partitions (3.37) of nominally equivalent volume, such that some partitions contain template and others do
not, followed by PCR amplification of target sequences and detection of specific PCR products, providing a
count of the number of partitions with a positive and negative signal for the target template
Note 1 to entry: dPCR can also provide the qualitative results “detected” or “not detected”.
Note 2 to entry: In certain instances, whole cells or organisms can be partitioned and lysis is performed in the
individual partitions to allow amplification of target templates.
[SOURCE: ISO 22174:2024, 3.7.1, modified — “amplicons” has been replaced by “PCR products”.]
3.17
deoxyribonucleic acid amplification
DNA amplification
multiplication of nucleic acid (3.31) through polymerase chain reaction (PCR) resulting in the amplified
PCR product
Note 1 to entry: The filling-in of the single-stranded DNA (3.12) by DNA polymerase (3.18) to double-stranded DNA,
starting from the primer (3.47) binding site, is also called elongation.
3.18
deoxyribonucleic acid polymerase
DNA polymerase
thermostable enzyme which catalyses DNA (3.12) synthesis
Note 1 to entry: DNA polymerase can also cleave a hybridized nucleic acid (3.31) molecule using its 5′-3′-exonuclease
activity (3.1). It is dependent on the type of enzyme and can be present in, for example, Taq-, Tth- and Tfl-polymerase.
Note 2 to entry: DNA synthesis is catalysed by reading the existing DNA sequence, then adding nucleotides to the newly
forming strand of DNA that are complementary to the original strand. By using a forward and a reverse primer (3.47),
DNA polymerases will duplicate both strands of the original source DNA resulting in two identical DNA molecules.
[SOURCE: ISO 22174:2024, 3.4.1, modified — Note 2 to entry has been added.]
3.19
eluate
solution obtained through nucleic acid extraction (3.32) and purification (3.33) that contains the nucleic
acids (3.31) of a sample and that is to be used for PCR
3.20
hybridization
specific binding of complementary nucleic acid sequences (3.34) under suitable reaction conditions
[SOURCE: ISO 22174:2024, 3.4.12]
3.21
intercalating dye
double-stranded DNA binding dye that emits fluorescence when bound to double-stranded DNA, that gets
also inserted into double-stranded DNA during the process of PCR amplification, increasing the intensity of
the fluorescence with the increase of double-stranded DNA
3.22
internal process control
control used for the quality assessment of the entire protocol, which is therefore added to the investigated
sample material to undergo the same procedure as the naturally present microorganism or genetic material
Note 1 to entry: Due to its mode of action, an internal control shall be selected which assumingly is naturally absent in
the tested matrix (3.25).
Note 2 to entry: For optimal use of the internal process control, it is recommended to use a control that behaves the
same way in all parts of the PCR-based method, e.g. a virus as a control for a virus PCR-based method and, in case of
bacteria, those that exhibit similar extraction behaviour (e.g. gram-positive or gram-negative).
[SOURCE: ISO 22174:2024, 3.5.3, modified — “target microorganism” has been replaced by “naturally
present microorganism or genetic material” and Note 2 to entry has been added.]
3.23
limit of blank
LoB
highest number of partitions (3.37) appearing positive, with more than
95 % probability, when testing samples in the absence of the target nucleic acid sequence (3.34) of the target
organism (3.66), which determines the target sequence specific “false positive” limit
Note 1 to entry: The LoB should be determined, as a minimum, from replicates of the negative amplification control
(3.5) (amplification LoB) (e.g. water, elution buffer) and negative samples containing the matrix (3.25) (full method
LoB). The number of negative control replicates and negative samples should be justified by the user laboratory and
should be consistent with the validation tests performed by the developer.
[SOURCE: ISO 22174:2024, 3.7.7, modified — “pathogen” has been replaced by “target organism”.]
3.24
master mix
mixture of reagents needed for nucleic acid (3.31) amplification except for the target nucleic acid sequence (3.34)
Note 1 to entry: A reverse transcription (RT)-master mix is a type of master mix that contains a mixture of reagents
needed for reverse transcription, at least consisting of a reverse transcriptase (3.58), a primer (3.47), deoxynucleotide
triphosphates (dNTPs) (3.14), ribonuclease (3.61) inhibitor and polymerase chain reaction-grade water (PCR-grade
water) (3.39).
Note 2 to entry: A PCR-master mix is a type of master mix that contains a mixture of reagents needed for DNA
amplification (3.17), at least consisting of a deoxyribonucleic acid (DNA)polymerase (3.18), primers, dNTPs and PCR-
grade water.
[SOURCE: ISO 22174:2024, 3.4.4, modified — Notes 1 and 2 to entry have been added.]
3.25
matrix
all the components of the sample
[SOURCE: ISO 22174:2024, 3.1.5]
3.26
multiplex polymerase chain reaction
multiplex PCR
PCR allowing the detection of multiple targets simultaneously within a single reaction tube, where more
primer (3.47) pairs [and probes (3.50)] are used within one master mix (3.24)
[SOURCE: ISO 22174:2024, 3.1.20]
3.27
negative cluster
set of negative results from partitions (3.37) that contained the reaction mix, without the target sequence,
representing the negative partitions
[SOURCE: ISO 22174:2024, 3.7.3]
3.28
negative extraction control
extraction blank
control carried through all steps of the nucleic acid extraction (3.32) procedure in the absence of a sample
[SOURCE: ISO 22174:2024, 3.5.4, modified — “test” has been deleted.]

3.29
negative polymerase chain reaction control
negative PCR control
no-template control
NTC
PCR control made with water (or other PCR-inert substrate such as grinding or elution buffer) free of target
nucleic acid (3.31) and PCR inhibitors
[SOURCE: ISO 22174:2024, 3.5.8]
3.30
negative process control
target free sample which is run through all stages o
...