ISO/TS 21085:2026

• Abstract:

  This document specifies requirements and considerations for handling, measuring, and storing samples with ultra-low concentration of target nucleic acid sequences, i.e., concentrations corresponding to copy numbers. This document is applicable to nucleic acid detection methods (qPCR, dPCR, isothermal amplification, and NGS).

• Status: Published
• Publication Date: 02-Aug-2026
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276 - Biotechnology
• Current Stage: 6060 - International Standard published
• Start Date: 03-Aug-2026
• Due Date: 08-Dec-2026
• Completion Date: 03-Aug-2026

Overview

ISO/TS 21085: Biotechnology - General requirements for the measurement of ultra-low concentration samples of target nucleic acid sequence is a technical specification developed by the International Organization for Standardization (ISO). This document sets forth essential requirements and best practices for handling, measuring, and storing ultra-low concentration nucleic acid sequence samples. These guidelines are highly relevant in applications utilizing nucleic acid amplification techniques such as quantitative PCR (qPCR), digital PCR (dPCR), isothermal amplification, and next-generation sequencing (NGS).

By addressing the unique challenges posed by the analysis of nucleic acid samples at extremely low copy numbers-where measurement results can be significantly impacted by stochastic (Poisson) distribution, adsorption to surfaces, and contamination-ISO/TS 21085 enables more reliable and accurate results in molecular biology laboratories, including those operating under ISO/IEC 17025.

Key Topics

• Ultra-low concentration definition: Outlines the challenges and requirements for samples containing target nucleic acids at levels (~3 to 100 copies/μL) where random distribution strongly influences results.
• Measurement and uncertainty: Standardizes units (copies, copies/μL, copies/well) and emphasizes the need to report measurement uncertainty, including contributions from both sample and process.
• Personnel competence: Highlights the requirement for specialized training and assessment of staff in areas including Poisson statistics, reference material handling, and contamination control.
• Labware and reagents: Details the importance of selecting low-adsorption, nuclease-free consumables and reagents, and includes guidance on avoiding loss of nucleic acids through adsorption, particularly to polypropylene or silica.
• Contamination control: Focuses on spatial and temporal separation of pre- and post-amplification steps, and proper cleaning protocols to prevent false results.
• Validation and verification: Describes methods for ensuring the reliability of results at ultra-low concentrations, including the use of certified reference materials, determination of limit of detection (LOD), dynamic range assessment, and robust reporting protocols.

Applications

ISO/TS 21085 provides critical standards for laboratories conducting ultra-sensitive nucleic acid testing in diverse fields:

• Clinical diagnostics: Detecting rare genetic variants, infectious agents, or cancer biomarkers in early disease screening where target DNA/RNA is present at extremely low levels.
• Environmental testing: Measuring trace amounts of environmental DNA (eDNA) for biodiversity, forensic, or contamination monitoring.
• Food safety and GMOs: Ensuring detection of genetically modified organisms at regulatory thresholds, or verifying the absence of prohibited DNA.
• Biotechnology research: Facilitating reproducibility and traceability in applications such as single cell genomics or studies involving low-input samples.

Implementing ISO/TS 21085 helps laboratories:

• Increase measurement confidence and data comparability
• Reduce variability and uncertainty associated with ultra-low copy detection
• Align processes with international best practices to support regulatory compliance and facilitate international trade

Related Standards

For laboratories integrating ISO/TS 21085, the following standards are particularly relevant:

• ISO 20395: Biotechnology - Requirements for evaluating the performance of quantification methods for nucleic acid target sequences (qPCR and dPCR)
• ISO/IEC 17025: General requirements for the competence of testing and calibration laboratories
• ISO 20070: Biotechnology - Biobanking - Requirements for biobanking of biological material
• ISO 21571: Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction

By adopting ISO/TS 21085 in conjunction with these standards, laboratories can further strengthen their capabilities in ultra-low nucleic acid analysis, ensuring the highest quality and integrity in measurement results.

Technical
Specification
ISO/TS 21085
First edition
Biotechnology — General
2026-08
requirements for the measurement
of ultra-low concentration samples
of target nucleic acid sequence
Biotechnologie — Exigences générales relatives au mesurage
des échantillons à très faible concentration de séquence d'acide
nucléide cible
Reference number
© ISO 2026
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
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or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
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Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
5 Requirements . 3
5.1 General .3
5.2 Unit .4
5.3 Personnel .4
5.4 Measurement environment .4
5.5 Equipment and reagents for measurements .5
5.5.1 General .5
5.5.2 Labware .5
5.5.3 Preventing contamination .5
5.6 Measurement method .5
5.6.1 Pre-analysis process .5
5.6.2 Measurement .6
5.6.3 Uncertainty .6
6 Validation and verification for ultra-low concentration sample of target nucleic acid
sequence . 7
6.1 Performance characteristics .7
6.1.1 General .7
6.1.2 Dynamic range .7
6.1.3 LOD .7
6.2 Performance evaluation of qPCR using NTC .9
6.3 Validation and verification .10
6.3.1 Validation .10
6.3.2 Verification .11
7 Reporting .11
Annex A (informative) Preparation of ultra-low concentration sample of target nucleic acid
sequence using gravimetric method .13
Annex B (informative) Evaluation of adsorption to labware using an instrument capable of
quantitatively evaluating macromolecular nucleic acids at ultra-low concentrations .15
Annex C (informative) Example of ultra-low concentration nucleic acid reference material . 17
Annex D (informative) Protocols for competency assessment using certified reference
materials . 19
Bibliography .21

iii
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
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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).
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This document was prepared by Technical Committee ISO/TC 276, Biotechnology, Subcommittee SC 1,
Analytical 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.

iv
Introduction
When measuring ultra-low concentration of nucleic acid samples, the measurements often exhibit significant
variation. This large variation is primarily caused by the Poisson distribution and other contributing
factors. In addition, the ultra-low concentration makes the samples highly susceptible to issues such as
adsorption and contamination. This document summarizes the key considerations and requirements for
handling, measurement including both qualification and quantification analysis, and storage of ultra-low
concentration sample of target nucleic acids sequence.
Analytical methods based on polymerase chain reaction (PCR) are theoretically capable of detecting the
presence of one molecule of nucleic acid in the reaction solution, making it possible to amplify and measure
the ultra-low concentration target nucleic acid sequence. These methods are used not only in the field of
biotechnology, but also in many other areas such as medicine, food, and the environment. However, it is
difficult to stably handle, measure, and store ultra-low concentration target nucleic acid sequences. For
example, nucleic acids readily adsorb to polypropylene (PP) materials, which is widely used as a base
material for labware, so target nucleic acid sequences stored in such containers are easily removed from
the sample/reaction solution during storage or experiment and become undetectable. Similarly, if a sample
before PCR and a sample after PCR are handled in the same place for the same target nucleic acid sequence,
contamination can occur, and the measurement result will change. Furthermore, when the target nucleic
acid sequence is present at ultra-low concentrations, the distribution of nucleic acid molecules becomes
non-uniform. These phenomena can occur even in samples of general concentrations, but the effects of
adsorption and contamination can be relatively small and negligible at higher concentrations. However,
since measurement results on ultra-low concentration samples can be impacted significantly, there are
special considerations to be taken into account when handing with such samples.
This document provides specific requirements to improve data reliability and obtain accurate measurement
results when working with ultra-low concentrations of target nucleic acid sequence. Biotechnology and
bioscience industry data with higher measurement confidence will enable data interoperability, improved
product quality, reduced risks and costs and facilitate international trade.

v
Technical Specification ISO/TS 21085:2026(en)
Biotechnology — General requirements for the measurement
of ultra-low concentration samples of target nucleic acid
sequence
1 Scope
This document specifies requirements and considerations for handling, measuring, and storing samples
with ultra-low concentration of target nucleic acid sequences, i.e., concentrations corresponding to copy
numbers.
This document is applicable to nucleic acid detection methods (qPCR, dPCR, isothermal amplification, and
NGS).
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 20395, Biotechnology — Requirements for evaluating the performance of quantification methods for nucleic
acid target sequences — qPCR and dPCR
3 Terms and definitions
For the purposes of this document, the following terms and definitions 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 https:// www .electropedia .org/
3.1
ultra-low concentration sample
[10]
sample containing 3 copies/μL to 100 copies/μL of target nucleic acid
sequences
Note 1 to entry: Ultra-low concentration sample of target nucleic acid sequence includes synthetized nucleic acid
solution diluted with TE buffer.
3.2
copy number
number of molecules (copies) containing a specific nucleic acid sequence
[SOURCE: ISO 20395:2019, 3.6]
3.3
copy number concentration
number of molecules (copies) containing a specific nucleic acid sequence in a defined volume
[SOURCE: ISO 20395:2019, 3.7]
3.4
negative nucleic acid target control
NTC
reference nucleic acid, or nucleic acid extracted from a certified reference material, or known negative
sample not containing the sequence under study
Note 1 to entry: This control demonstrates that the results of analyses of test samples not containing the target
sequence will be negative.
[SOURCE: ISO 24276:2006, 3.4.2; modified — DNA replaced by nucleic acid.]
3.5
limit of detection
LOD
measured quantity value, obtained by a given measurement procedure, for which the probability of falsely
claiming the absence of a component in a material is β, given a probability α of falsely claiming its presence
[SOURCE: ISO 20395:2019, 3.14]
3.6
probability of detection
POD
proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte
level or concentration
Note 1 to entry: For qualitative methods, POD represents the probability of detection.
[SOURCE: ISO 16140-4:2020, 3.9]
3.7
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended use
or application have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.13, modified — Notes to entry deleted.]
3.8
verification
confirmation, through the provision of objective evidence, that specified requirements have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.12, modified — Notes to entry deleted.]
3.9
precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
[SOURCE: ISO/IEC Guide 99:2007, 2.15, modified — Notes to entry deleted.]
3.10
repeatability
measurement precision under a set of repeatability conditions of measurement
[SOURCE: ISO/IEC Guide 99:2007, 2.21]
3.11
intermediate precision
measurement precision under a set of intermediate precision conditions of measurement
[SOURCE: ISO/IEC Guide 99:2007, 2.23, modified — Note to entry deleted.]

3.12
reproducibility
measurement precision under reproducibility conditions of measurement
[SOURCE: ISO/IEC Guide 99:2007, 2.25, modified — Note to entry deleted.]
3.13
measurement uncertainty
non-negative parameter characterizing the dispersion of the quantity values being attributed to a
measurand, based on the information used
[SOURCE: ISO/IEC Guide 99:2007, 2.26, modified — Notes to entry deleted.]
3.14
robustness
measure of a test method's capacity to remain unaffected by small, but deliberate variations in method
parameters and provides an indication of its reliability during normal usage
[SOURCE: ISO 23033:2021 3.44]
3.15
test sample
sample prepared for testing or analysis, the whole quantity or part of it being used for testing or analysis at
one time
[SOURCE: ISO 16577:2016, 3.210]
4 Principle
In this document, the following two patterns are assumed for the ultra-low concentration sample of target
nucleic acid sequence.
a) Samples with ultra-low concentrations of both the matrix-derived nucleic acids and the target nucleic
acid sequence.
EXAMPLE 1 Environmental DNA, NA derived from highly processed food, samples in forensic science, fossil,
series of standard for the validation and the verification without carrier NA.
b) Samples with an ultra-low concentration of the target of nucleic acid sequence and a high concentration
of matrix-derived nucleic acids.
EXAMPLE 2 GMO, samples in medical laboratory, microbiology.
When considering the sample specified in 4 a), the effects of the adsorption can be significant, while the effect
of the adsorption can be negligible for the sample specified in 4 b). Variation due to Poisson distribution and
other factors can affect both samples.
5 Requirements
5.1 General
There are several points to consider for the measurement of ultra-low concentration samples of target
nucleic acid sequence compared to the measurement for those of general concentrations. In addition, since
the impact of contamination is relatively greater in ultra-low concentration nucleic acid measurements than
in general-concentration nucleic acid measurements, efforts to prevent contamination shall be implemented
according to the purpose of the measurement. Examples of specific points to consider are described in the
relevant subclauses.
5.2 Unit
Mass (g, ng, etc.) or mass concentration (g/mL, ng/μL, etc.) is usually used as a unit to indicate the amount
of nucleic acid sample, however the amount of the target nucleic acid sequence contained in an ultra-low
concentration sample of target nucleic acid sequence is not appropriately expressed as a mass.
In order to clearly indicate the amount of the target nucleic acid sequence contained, units focusing on the
number of target nucleic acid sequence molecules shall be used, for example, copy number (copies), copy
number per well (copies/well), copy number concentration (copies/μL, copies/mL).
NOTE Spectrophotometric methods primarily measure the mass or mass concentration of nucleic acids, but are
not applied to measure single molecules. Methods that detect single molecules are better suited to quantify ultra-low
concentration sample of target nucleic acid sequence.
5.3 Personnel
Personnel supervising the measurement of ultra-low concentration sample of target nucleic acid sequence
require additional competence beyond that required for measurements in the normal concentration range.
Since the measurement of ultra-low concentration sample of target nucleic acid sequence requires special
knowledge and skills, a training plan for personnel shall be developed.
Indicators for evaluating the competence of personnel shall be established. Confirmatory testing using
uniform samples and protocols should be performed to evaluate the competence of personnel. The samples
used should be reference materials with assigned concentrations, preferably certified reference materials.
An example of confirmation test method using reference materials is given in Annex D.
Personnel shall be trained according to the type of work to be performed to acquire the necessary and
sufficient knowledge on handling, storing, and measuring ultra-low concentration sample of target nucleic
acid sequence.
The educational program can include, but is not limited to the following knowledge:
— Knowledge of statistics: Poisson distribution;
— Metrological knowledge;
— Knowledge of dispensing methods;
— Knowledge of reference materials;
— Knowledge of physical properties of materials and samples;
— Knowledge of physical properties of nucleic acids: adsorption, limit of detection (LOD), probability of
detection (POD), etc;
— Content of related standards.
NOTE Further general guidance can be found in References [5], [6].
5.4 Measurement environment
Sample contamination with nucleic acids from extraneous sources can occur. When handling ultra-low
concentration sample of target nucleic acid sequence, even very small amounts of contaminants, especially
those with the same or similar sequences as the target, can significantly affect the measurement results.
Therefore, steps shall be taken to minimise contamination in the ultra-low concentration sample of target
nucleic acid sequence.
Incompatible operations should be separated spatially or temporally. In particular, it is required to handle
pre-PCR and post-PCR samples in different areas or compartments.

After the PCR is completed, the samples shall be discarded without opening the tube lid or removing the plate
seal, except when performing the POST-PCR methods. When performing the POST-PCR methods including
electrophoresis, restriction enzyme treatment, cloning, POST-PCR sequencing (e.g. NGS), or analysis of
reaction products by other methods, the sample shall be opened in a different area or compartments.
If an accident such as leakage occurs during/after the PCR reaction, cleaning and decontamination shall be
performed.
NOTE Cleaning with an approximate 5 % sodium hypochlorite solution can be effective.
5.5 Equipment and reagents for measurements
5.5.1 General
The equipment and reagents for the measurement shall be selected with consideration for measuring the
ultra-low concentration sample of nucleic acid sequences. Labware shall be nuclease-free and with low
adsorption for nucleic acids.
5.5.2 Labware
In relation to the sample described in 4a):
Labware, such as tube and micropipette tips, that directly contact with nucleic acid samples should be
selected in consideration of adsorption. Since nucleic acids are adsorbed especially to polypropylene and
silica, when handling the sample contains ultra-low concentration sample of target nucleic acid sequence
and matrix NA, they can be lost due to adsorption on the labware. An example of the verification method is
shown in Annex B. It should also be evaluated that eluates from the labware surface do not affect the results.
Since the presence of a large amount of nucleic acids other than the target nucleic acid sequence can reduce
adsorption of the target nucleic acid sequence to the labware, carrier nucleic acids may be used as needed.
[2]
NOTE Detailed information on containers, such as tubes, can be found in ISO 20070 .
5.5.3 Preventing contamination
In relation to both samples described in 4a) and 4b):
It is required to develop a measurement environment that reduces the risk of contamination of nuclease.
Equipment shall be cleaned regularly to prevent contamination of nuclease. When handling RNA, reagents
shall be RNase free.
Reagents expected to be used more than once, for example primers, probes, master mixes, H O and TE shall
be handled in a different area/time from the sample containing the target nucleic acid because they can easily
become contaminated. It is recommended to aliquot these reagents to reduce introduction of contaminants.
Reagents shall be stored with clear identification, for example the name and date of preparation.
The use of a master mix containing Uracil-DNA Glycosylase (UNG) and dUTP can be helpful in PCR to prevent
contamination due to carryover.
5.6 Measurement method
5.6.1 Pre-analysis process
During sample preparation, the target nucleic acid sequence can be also degraded and adsorbed. The sample
shall be handled with the same attention to contamination as when preparing reagents (5.5).
In an ultra-low concentration sample of target nucleic acid sequence, it is difficult to prepare the sample to
the desired concentration or number of molecules. This is generally attributed to the significant influence
[16]
of the Poisson distribution, although other factors can also play a role. Therefore, the number of steps for
sample preparation should be minimized.

An example of sample preparation using the gravimetric method is shown in Annex A.
The nucleic acid extraction method should be selected in a manner that has minimal impact on the
subsequent measurement process. In the performance evaluation of the extraction process, the use of a
reference material with a known concentration allows a recovery test to be performed.
[3]
NOTE ISO 21571 can be referred .
When the measurement target is RNA, reverse transcription reaction is required. Since the efficiency of the
reverse transcription reaction affects the measurement results, the conditions of the reverse transcription
reaction shall be documented. If detailed optimization is performed, the method of optimization shall also
be documented. The reverse transcription efficiency can also be evaluated by adding the reference materials
to the same matrix as the sample and performing the reverse transcription reaction process.
The extraction and reverse transcription reaction steps can be evaluated separately from the subsequent
sample preparation and PCR performance evaluation. Performance evaluation should be made on samples
that are close to the actual sample.
5.6.2 Measurement
5.6.2.1 General
Examples of methods for measuring ultra-low concentration sample of target nucleic acid sequence include
qPCR, dPCR, isothermal amplification, and NGS. Each method shall be evaluated for sufficient performance
and validity to measure ultra-low concentration sample of target nucleic acid sequence.
When preparing a concentration series of a sample, the number of molecules in the aliquots used to prepare
the concentration series is significantly affected by various factors. The influence of these factors on the
preparation of the desired concentration series shall be considered to select a suitable number of replicate
measurements.
5.6.2.2 qPCR
When measuring ultra-low concentration sample of target nucleic acid sequence using qPCR, the C value
q
obtained as a result of the measurement is expected to be high. To ensure that the C value is correct, the
q
performance evaluation and validation shall be performed. The methods of performance evaluation and
validation are described in 6.2. A reference material including an in-house sample and a spiked sample, with
known concentration of the same sequence as the target nucleic acid sequence shall be used for performance
evaluation and validation. When in-house or spiked samples are used, they shall be validated.
5.6.2.3 dPCR
When measuring ultra-low concentration sample of target nucleic acid sequence using dPCR, the quantifiable
concentration range (dynamic range) shall be identified using an ultra-low concentration standard. Normally,
dPCR measures a sample stock solution at an ultra-low concentration, and if it is within the quantifiable
concentration range, the measured value at that time can be adopted. A reference material with the same
sequence as the target nucleic acid sequence should be used for performance evaluation and validation.
When in house sample or spiked sample are used instead, they should be validated.
5.6.3 Uncertainty
Measurement uncertainty associated with the result can have contributions from uncertainties in counting
the target nucleic acid molecules and from determination of the amount of sample analysed (usually the
sample volume). The uncertainty of the measured concentration of target nucleic acid should be given. In
addition, the examination uncertainty for proper identification of the target nucleic acid sequence should be
given.
6 Validation and verification for ultra-low concentration sample of target nucleic
acid sequence
6.1 Performance characteristics
6.1.1 General
For measurements at ultra-low concentration sample of target nucleic acid sequence, both quantitative and
qualitative analyses shall be evaluated for performance and validation for the intended use.
In qualitative analysis, the detectable range shall be determined. Determination of the detectable range
requires the use of appropriate reference materials and the determination of dynamic range and LOD.
In quantitative analysis, the quantifiable range shall be determined. Determination of the quantifiable range
requires the determination of a confidence interval for quantifiability by creating a calibration curve using
appropriate reference materials.
6.1.2 Dynamic range
When using either qualitative analysis in qPCR or quantitative analysis in qPCR/dPCR, the possible
qualitative/quantitative range shall be determined using a concentration series of test samples prepared
from a reference material to which a concentration value is assigned. The reference materials used in this
process should be certified reference materials.
For qualitative analysis,
...