SIST EN ISO 21286:2020

Soil quality - Identification of ecotoxicological test species by DNA barcoding (ISO 21286:2019)

This document specifies a protocol to identify ecotoxicological test specimens (mainly invertebrates and plants) to the species level, based on the DNA barcoding technique. This protocol can be used by laboratories performing DNA barcoding in order to standardize both the wet-lab and data analysis workflows as much as possible, and make them compliant with community standards and guidelines.
This document does not intend to specify one particular strain for each test method, but to accurately document the species/strain which was used.
NOTE 1    This does not imply that DNA barcoding is performed in parallel to each test run, but rather regularly (e.g. once a year, such as reference substance testing) and each time a new culture is started or new individuals are added to an ongoing culture.
This document does not aim at duplicating or replacing morphological-based species identifications. On the contrary, DNA barcoding is proposed as a complementary identification tool where morphology is inconclusive, or to diagnose cryptic species, in order to ensure that the results obtained from different ecotoxicological laboratories are referring to the same species or strain.
This document is applicable to identifications of immature forms which lack morphological diagnostic characters (eggs, larvae, juveniles), as well as the streamline identification of specimens collected in field monitoring studies, where large numbers of organisms from diverse taxa are classified.
NOTE 2    In principle, all species regularly used in ecotoxicological testing can be analysed by DNA barcoding. Besides the earthwoms Eisenia fetida and E. andrei, further examples for terrestrial species are Lumbricus terrestris, L. rubellus, Allolobophora chlorotica, Aporrectodea rosea, and A. caliginosa, Dendrodrilus rubidus, Enchytraeus albidus, and E. crypticus (Haplotaxida); Folsomia candida, F. fimetaria, Proisotoma minuta, and Sinella curviseta (Collembola); Hypoaspis aculeifer and Oppia nitens (Acari); Aleochara bilineata and Poecilus cupreus (Coleoptera); Scathophaga stercoraria, Musca autumnalis (Diptera) or Pardosa sp. (Arachnida). Nematodes or snails and even plants can also be added to this list.

Bodenbeschaffenheit - Identifizierung der Testorganismenarten für ökotoxikologische Tests mit Hilfe von DNA-Barcoding (ISO 21286:2019)

Dieses Dokument beschreibt ein Protokoll für die Identifizierung ökotoxikologischer Testorganismen (hauptsächlich Invertebraten und Pflanzen) auf Artenebene mit Hilfe des DNA Barcodings. Dieses Protokoll kann von Labors verwendet werden, die das DNA Barcoding für die weitgehende Standardisierung des Labor  und Datenanalyse Workflows einsetzen, damit diese Labors im Einklang mit üblichen Standards und Leitfäden arbeiten.
Dieses Dokument soll keinen bestimmten Stamm für jedes Prüfverfahren, sondern das präzise Dokumentieren der verwendeten Arten/Stämme vorgeben.
ANMERKUNG 1 Dies bedeutet nicht, dass das DNA Barcoding parallel zu jedem Prüfungsdurchgang durchgeführt werden muss, sondern nur regelmäßig (z. B. einmal jährlich, zum Beispiel bei Prüfungen von Referenzstoffen) und immer, wenn eine neue Kultur angelegt oder einer vorhandenen Kultur neue Individuen hinzugefügt werden.
Dieses Dokument hat nicht das Ziel, die morphologisch basierte Identifizierung von Arten zu kopieren oder zu ersetzen. Das DNA Barcoding wird vielmehr als ein ergänzendes Instrument zur Identifizierung vorgeschlagen, wenn die Morphologie keine schlüssigen Ergebnisse liefert oder für die Diagnose kryptischer Arten. Damit wird sichergestellt, dass die Ergebnisse verschiedener Ökotoxikologie Labors sich auf die gleiche Art oder den gleichen Stamm beziehen.
Dieses Dokument ist anwendbar für die Identifizierung unreifer Formen ohne morphologische Diagnosemerkmale (Eier, Larven, Jugendstadium), sowie für die durchgängige Identifizierung von Proben, die in Feldüberwachungsstudien gesammelt wurden und bei denen zahlreiche Organismen unterschiedlicher Taxa klassifiziert werden.
ANMERKUNG 2 Grundsätzlich können alle Arten, die üblicherweise für ökotoxikologische Prüfungen verwendet werden, durch DNA Barcoding analysiert werden. Neben den Regenwürmern Eisenia fetida und E. andrei sind weitere Beispiele landbewohnender Arten Lumbricus terrestris, L. rubellus, Allolobophora chlorotica, Aporrectodea rosea und A. caliginosa, Dendrodrilus rubidus, Enchytraeus albidus und E. crypticus (Haplotaxida); Folsomia candida, F. fimetaria, Proisotoma minuta und Sinella curviseta (Collembola); Hypoaspis aculeifer und Oppia nitens (Acari); Aleochara bilineata und Poecilus cupreus (Coleoptera); Scathophaga stercoraria, Musca autumnalis (Diptera) oder Pardosa sp. (Arachnida). Nematoden oder Schnecken und sogar Pflanzen können dieser Liste auch hinzugefügt werden.

Qualité du sol - Identification des espèces par codes-barres ADN dans les essais d'écotoxicologie (ISO 21286:2019)

Le présent document spécifie un protocole d'identification de spécimens d'essais écotoxicologiques (principalement des invertébrés et des végétaux) au niveau de l'espèce, reposant sur la technique du code-barres ADN. Ce protocole peut être utilisé par les laboratoires effectuant le code-barres ADN afin de normaliser le plus possible les travaux de laboratoire et les flux d'analyse de données, et de les mettre en conformité avec les normes et les lignes directrices communautaires.
Le présent document ne prévoit pas de spécifier une souche particulière pour chaque méthode d'essai, mais de documenter avec exactitude l'espèce/la souche qui a été utilisée.
NOTE 1    Cela ne veut pas dire que le code-barres ADN est effectué parallèlement à chaque cycle d'essai, mais qu'il est effectué régulièrement (par exemple, une fois par an, notamment pour l'essai mené avec la substance de référence) et à chaque fois qu'une nouvelle culture est démarrée ou que de nouveaux individus sont ajoutés à une culture existante.
Le présent document ne vise pas à reproduire ou remplacer les identifications d'espèces reposant sur des caractéristiques morphologiques. En revanche, le code-barres ADN est proposé comme outil d'identification complémentaire lorsque l'identification morphologique est incertaine, ou pour diagnostiquer les espèces cryptiques, afin d'assurer que les résultats obtenus auprès de différents laboratoires d'écotoxicologie font référence à la même espèce ou souche.
Le présent document est applicable à l'identification de formes immatures n'ayant pas de caractéristiques morphologiques de diagnostic (œufs, larves, juvéniles) ainsi qu'à l'identification rationalisée des spécimens prélevés lors d'études de surveillance sur le terrain, où un grand nombre d'organismes de taxons divers sont classés.
NOTE 2    En principe, toutes les espèces régulièrement utilisées lors des essais écotoxicologiques peuvent être analysées par code-barres ADN. Outre les vers de terre Eisenia fetida et E. andrei, d'autres exemples d'espèces terrestres sont Lumbricus terrestris, L. rubellus, Allolobophora chlorotica, Aporrectodea rosea, A. caliginosa, Dendrodrilus rubidus, Enchytraeus albidus et E. crypticus (Haplotaxida) ; Folsomia candida, F. fimetaria, Proisotoma minuta et Sinella curviseta (Collembola) ; Hypoaspis aculeifer et Oppia nitens (Acari) ; Aleochara bilineata et Poecilus cupreus (Coleoptera) ; Scathophaga stercoraria, Musca autumnalis (Diptera) ou Pardosa sp. (Arachnida). De plus, les nématodes ou les escargots et même les plantes peuvent être ajoutés à cette liste non exhaustive.

Kakovost tal - Identifikacija vrst preskusnih organizmov za ekotoksikološke preskuse s črtnim kodiranjem DNK (ISO 21286:2019)

General Information

• Status: Published
• Public Enquiry End Date: 02-Feb-2020
• Publication Date: 23-Sep-2020
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: KAT - Soil quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 17-Aug-2020
• Due Date: 22-Oct-2020
• Completion Date: 24-Sep-2020
• Ref Project: EN ISO 21286:2020 - Soil quality - Identification of ecotoxicological test species by DNA barcoding (ISO 21286:2019)

Overview

EN ISO 21286:2020 (ISO 21286:2019) - published by CEN/ISO - establishes a standardized protocol for identifying ecotoxicological test species by DNA barcoding. The standard applies to soil quality testing and specifies both wet‑lab and data‑analysis workflows so laboratories can achieve reproducible, comparable species‑level identifications. It is intended as a complementary tool to morphological taxonomy, particularly useful when morphology is inconclusive, for diagnosing cryptic species, or for identifying immature forms (eggs, larvae, juveniles) and bulk field samples.

Key Topics

• Scope and intent: Standardizes DNA barcoding for ecotoxicological test specimens (mainly invertebrates and plants) without prescribing specific strains - requires accurate documentation of the species/strain used.
• Workflow elements: Covers biological material, reagents, apparatus, contamination avoidance and safety precautions.
• Laboratory procedures: Defines the main steps - DNA isolation, quantification, PCR (including target genomic regions and primer design/synthesis), amplicon checking, purification and sequencing.
• Bioinformatics & data quality: Includes guidance on electropherogram/raw sequence quality checking, trimming low‑quality regions and primers, sequence overlap and editing, sequence verification, species assignment and the importance of reference database quality.
• Reporting & QA: Requirements for calculation and expression of results, validity of the test and content of test reports to support traceability and inter‑laboratory comparability.
• Operational guidance: Recommends routine re‑identification (e.g., regularly such as annually or when new cultures are started) rather than per‑run barcoding.

Applications

• Ecotoxicology laboratories seeking consistent species identification for toxicity and reproduction tests.
• Environmental testing and monitoring programs that process large numbers of mixed taxa from soil or field samples.
• Research groups and culture collections documenting strain identity and avoiding cryptic species or culture mixing.
• Regulators and QA managers who require documented, reproducible species identity to improve test comparability and data reliability.

Practical benefits include improved quality assurance, reduced taxonomic uncertainty (e.g., Eisenia fetida/E. andrei complexes), and better comparability of ecotoxicological results across laboratories.

Related Standards

• ISO 11268‑1, ISO 11268‑2, ISO 17512‑1 (earthworm and soil organism test methods referenced in the standard)
• ISO 11267, ISO 17512‑2 (springtail and related ecotoxicological methods)

Keywords: EN ISO 21286:2020, ISO 21286:2019, DNA barcoding, soil quality, ecotoxicological test species, species identification, molecular identification, laboratory standard, CEN.

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2020
Kakovost tal - Identifikacija vrst preskusnih organizmov za ekotoksikološke
preskuse s črtnim kodiranjem DNK (ISO 21286:2019)
Soil quality - Identification of ecotoxicological test species by DNA barcoding (ISO
21286:2019)
Bodenbeschaffenheit - Identifizierung der Testorganismenarten für ökotoxikologische
Tests mit Hilfe von DNA-Barcoding (ISO 21286:2019)
Qualité du sol - Identification des espèces par codes-barres ADN dans les essais
d'écotoxicologie (ISO 21286:2019)
Ta slovenski standard je istoveten z: EN ISO 21286:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 21286
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Identification of ecotoxicological test species
by DNA barcoding (ISO 21286:2019)
Qualité du sol - Identification des espèces par codes- Bodenbeschaffenheit - Allgemeine Anleitung zur
barres ADN dans les essais d'écotoxicologie (ISO Verwendung des DNA-Barcodes in ökotoxikologischen
21286:2019) Untersuchungen (ISO 21286:2019)
This European Standard was approved by CEN on 13 April 2020.

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
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21286:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 21286:2019 has been prepared by Technical Committee ISO/TC 190 "Soil quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 21286:2020 by
Technical Committee CEN/TC 444 “Environmental characterization of solid matrices” the secretariat of
which is held by NEN.
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 October 2020, and conflicting national standards shall
be withdrawn at the latest by October 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 21286:2019 has been approved by CEN as EN ISO 21286:2020 without any modification.

INTERNATIONAL ISO
STANDARD 21286
First edition
2019-03
Soil quality — Identification of
ecotoxicological test species by DNA
barcoding
Qualité du sol — Identification des espèces par code-bare ADN dans
les essais d'écotoxicologie
Reference number
ISO 21286:2019(E)
©
ISO 2019
ISO 21286:2019(E)
© 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
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CH-1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 21286:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and material . 3
5.1 Biological material . 3
5.2 Enzyme . 3
5.3 Oligonucleotide PCR primers . 3
5.4 Reagents. 3
6 Apparatus . 4
7 General requirements . 5
7.1 Experimental precaution and contamination avoidance . 5
7.2 Safety precautions . 5
7.2.1 Chemical hazards. 5
7.2.2 Physical hazards . 6
8 Procedure. 6
8.1 DNA isolation . 6
8.2 Quantification . 7
8.3 PCR . 7
8.3.1 Target genomic region . 7
8.3.2 Primer design . 7
8.3.3 Primer synthesis . 7
8.3.4 PCR . 8
8.4 Checking the amplicon size . 9
8.5 Purification . 9
8.6 Sequencing . 9
8.7 Bioinformatics . 9
8.7.1 General. 9
8.7.2 Electropherogram or raw sequence quality checking .10
8.7.3 Trimming of low-quality regions and primers sequences .10
8.7.4 Sequence overlapping .10
8.7.5 Sequence verification . .11
8.7.6 Reviewing the edited sequence .11
8.7.7 Species assignment .11
8.7.8 Quality of the reference databases .12
9 Calculation and expression of results .13
10 Validity of the test .13
11 Test report .14
Annex A (informative) Eisenia Barcoding Initiative: A ring test to evaluate the applicability
of DNA barcoding for the identification of Eisenia species .15
Bibliography .18
ISO 21286:2019(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 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 190, Soil quality, Subcommittee SC 4,
Biological characterization.
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

ISO 21286:2019(E)
Introduction
Currently, test species identification is usually based on morphological characters. However, this does
not always give clear results because
a) few taxonomic experts are available,
b) closely related species can differ by a few, easily overlooked characters, and
c) even more importantly, several test species are in fact complexes of cryptic species.
A good example is the compost worm Eisenia fetida/andrei (used in ISO 11268-1, ISO 11268-2 and
ISO 17512-1), in which morphological traits alone may not be sufficient to discriminate between both
[5][36] [50]
species . Another well-known case is the predatory mite, Hypoaspis (Geolaelaps) aculeifer ,
[31]
which might get confused with H. miles, widely used in biological pest control .
Species misidentifications, the use of a morphospecies which is actually a complex of cryptic species, or
even species mixing in lab cultures, can be a serious problem for the reliability of the ecotoxicological
tests. Sibling species in a morphospecies complex can exhibit ecological, behavioural, and physiological
differences, and can differ also in their response to toxicants (e.g. References [2], [17], [35], [40]). This
also seems to be the case of the springtail Folsomia candida (used in ISO 11267 and ISO 17512-2), in
which considerable levels of genetic differentiation have been found among natural populations of F.
[9][19][41]
candida and among laboratory strains . Although different laboratory strains have been found
[12][9]
to exhibit only minor differences in the sensitivity towards some chemicals , other studies have
detected significant variation in phenmedipham avoidance behaviour and divergent fitness responses
[14][30]
to cadmium exposure among genetically differentiated strains . Moreover, even if two species
have similar responses to toxicants, the presence of two species within the same laboratory culture can
[36]
result in the production of sterile hybrids, which will bias the outcome of reproduction tests .
Implementing species identification via DNA barcoding can help to overcome these obstacles, ensuring
that the species or strain used for testing is well characterized. As a result, quality assurance can be
improved, making the results obtained by different ecotoxicological laboratories far more reliable and
comparable. For Eisenia fetida/E. andrei this work, including an international ringtest, has already been
[36]
performed , see Annex A. The conclusions of this ringtest can be summarized as follows.
— DNA barcoding is a reliable and practical method for identifying Eisenia species.
— Only 17 out of 28 ecotoxicological laboratories were correct in their taxonomic assignment. Most
laboratories with wrong or unknown assignments actually have E. andrei in stock.
— The existence of a cryptic species pair within E. fetida is a plausible hypothesis.
— It is important that earthworms used for ecotoxicological tests are regularly (re-)identified by DNA
barcoding.
Very probably, similar experiences and recommendations can be drawn for other invertebrates
species used in terrestrial ecotoxicology, as well as plants. Indeed, DNA barcoding has proven to be
useful for specimen identification and species delimitation in many organism groups, including other
[13][37] [16] [15] [32] [42] [28]
earthworms , enchytraeids , mites , collembolans , molluscs , nematodes and
[8]
terrestrial plants .
INTERNATIONAL STANDARD ISO 21286:2019(E)
Soil quality — Identification of ecotoxicological test species
by DNA barcoding
1 Scope
This document specifies a protocol to identify ecotoxicological test specimens (mainly invertebrates
and plants) to the species level, based on the DNA barcoding technique. This protocol can be used by
laboratories performing DNA barcoding in order to standardize both the wet-lab and data analysis
workflows as much as possible, and make them compliant with community standards and guidelines.
This document does not intend to specify one particular strain for each test method, but to accurately
document the species/strain which was used.
NOTE 1 This does not imply that DNA barcoding is performed in parallel to each test run, but rather regularly
(e.g. once a year, such as reference substance testing) and each time a new culture is started or new individuals
are added to an ongoing culture.
This document does not aim at duplicating or replacing morphological-based species identifications. On
the contrary, DNA barcoding is proposed as a complementary identification tool where morphology is
inconclusive, or to diagnose cryptic species, in order to ensure that the results obtained from different
ecotoxicological laboratories are referring to the same species or strain.
This document is applicable to identifications of immature forms which lack morphological diagnostic
characters (eggs, larvae, juveniles), as well as the streamline identification of specimens collected in
field monitoring studies, where large numbers of organisms from diverse taxa are classified.
NOTE 2 In principle, all species regularly used in ecotoxicological testing can be analysed by DNA barcoding.
Besides the earthwoms Eisenia fetida and E. andrei, further examples for terrestrial species are Lumbricus
terrestris, L. rubellus, Allolobophora chlorotica, Aporrectodea rosea, and A. caliginosa, Dendrodrilus rubidus,
Enchytraeus albidus, and E. crypticus (Haplotaxida); Folsomia candida, F. fimetaria, Proisotoma minuta, and Sinella
curviseta (Collembola); Hypoaspis aculeifer and Oppia nitens (Acari); Aleochara bilineata and Poecilus cupreus
(Coleoptera); Scathophaga stercoraria, Musca autumnalis (Diptera) or Pardosa sp. (Arachnida). Nematodes or
snails and even plants can also be added to this list.
2 Normative references
There are no normative references in this document.
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 http: //www .electropedia .org/
3.1
amplicon
specific DNA product generated by PCR (3.5) using one pair of PCR primers (3.6)
3.2
DNA barcode
unique pattern of DNA sequence that identifies each species
ISO 21286:2019(E)
3.3
electropherogram
trace file
combination of a graphical representation of a Sanger DNA sequence composed of colour-coded peaks
with each colour corresponding to one nucleotide
Note 1 to entry: They are automatically supplied by DNA sequencing programs.
3.4
Phred quality score
Q score
quality measure used to assess the accuracy of a sequencing reaction
Note 1 to entry: This quality measure indicates the probability that a given base is called incorrectly by the
sequencer. Phred scores are on a logarithmic scale. Therefore, if Phred assigns a Q score of 30 (Q30) to a base, this
is equivalent to the probability of an incorrect base call 1 in 1 000 times. A lower base call accuracy of 99 % (Q20)
will have an incorrect base call probability of 1 in 100, meaning that every 100 base pairs sequencing read will
likely contain an error.
3.5
polymerase chain reaction
PCR
molecular biology technique for rapidly synthesising multiple copies of a given DNA segment by using a
DNA polymerase and an oligonucleotide primer pair
3.6
PCR primer
short oligonucleotides (usually 15 to 30 nucleotides in length) that allow PCR amplification of DNA
between specific sites
Note 1 to entry: The two primers (a forward and a reverse) are base-paired to the top and bottom strand of the
template DNA, and their 3’-OH ends are in convergent direction.
4 Principle
DNA barcoding is a molecular method that uses a short and standardized DNA region (the DNA barcode)
[22]
as a genetic tag for species-level identification .
Since its inception in 2003 and the launch of the Barcode of Life project, DNA barcoding has
systematically been applied not only to biological research, but also to several industrial fields where
a correct identification of biological materials is essential, such as the food industry. For example, it
[29]
is helping to detect fraud in herbal medicinal products , and it has been adopted by the Food and
[21][44]
Drug Administration (FDA) for seafood and fish identification . In fact, DNA barcoding is likely to
[20]
become a routine test in many fields, in particular in food quality control and traceability .
Briefly, the goal of DNA barcoding is:
a) to obtain the nucleotide sequence of a standardised DNA region from an unidentified sample (a test
specimen),
b) to compare that sequence with known sequences in a reference database by using bioinformatic
methods, and
c) based on such comparison, to identify the sample to the species level.
Therefore, DNA barcoding cannot be a useful identification tool without a reliable and comprehensive
reference database, which includes enough samples of each species from across its geographic range to
account for intraspecific variability. Also, DNA barcoding relies on the premise that sequences in this
barcode region are more similar between members of a species than to sequences of any other species
(the so called barcode gap). Therefore, before applying DNA barcoding, a species delimitation study
2 © ISO 2019 – All rights reserved

ISO 21286:2019(E)
of the target organismal group should have been carried out to assess its efficacy for discriminating
species.
It is essential that the DNA barcoding method is carried out by trained staff. On the one hand, trained
laboratory technicians are needed to optimize the wet-lab protocols for each organismal group.
On the other hand, the wet-lab pipeline needs to be supervised by scientists trained in genomics
and systematics. These scientists should also be in charge of the electropherogram and/or raw DNA
sequence file analysis and species assignment.
5 Reagents and material
5.1 Biological material
Adequate specimen preservation is a critical factor to obtain good-quality DNA from samples. Whenever
possible, specimen samples for DNA barcoding should be taken from freshly harvested or fresh-frozen
tissue. Exposure to preservation agents such as ethyl acetate or formaldehyde should be avoided, as
they destroy DNA.
Freezing at –80 °C or in liquid nitrogen (−196 °C) is the preferred method for long-term storage of
tissue samples. DNA in dried specimens generally remains stable for at least one year, but degradation
[23]
becomes increasingly problematic over time .
Ethanol-preserved material is easily analysed when fresh, but DNA will slowly become acidified and
degraded unless ethanol is regularly refreshed or buffered. For proper tissue preservation, use an
ethanol concentration of 95 % to 99 %, and ensure that the volume of ethanol is at least three times
greater than the volume of tissue. In order to maintain the ethanol concentration to at least 95 %, it is
necessary to replace the ethanol solution within the first days (at least three days) after sampling, and
[23]
tightly seal the vial to avoid evaporation . A combination of low temperatures (–20 °C) and ethanol
will help preserve the samples for long-term storage and helps prevent degradation during thawing and
re-freezing cycles.
As a general rule, DNA barcoding analysis should follow tissue collection as soon as possible, but specimens
[21][23]
adequately preserved and stored for several months will perform well in DNA extraction .
5.2 Enzyme
Taq Polymerase from Thermus aquaticus is standard for PCR. Hot start Taq polymerases and/or high
fidelity DNA polymerases have been shown to offer a high performance in DNA barcoding, allowing
for greater amplification sensitivity and increased ease of reaction setup than standard polymerases
(http: //ccdb .ca/resources/).
Alternatively, pre-optimised commercial master mixes may be used. These consist of a premixed,
ready-to-use solution containing Taq DNA polymerase, dNTPs, MgCl and reaction buffers at optimal
concentrations for efficient amplification of DNA templates in routine PCR.
5.3 Oligonucleotide PCR primers
For Oligonucleotide PCR primers, see 8.3.2 and 8.3.3.
5.4 Reagents
5.4.1 Nuclease-free water molecular grade water (dd H O).
5.4.2 TE buffer (Tris-EDTA buffer), 1-fold, pH 8,0.
Dissolve 1 ml of 1 mol/l Tris base (pH 8,0), 0,2 ml EDTA (0,5 mol/l) in 98,8 ml of molecular grade water.
Adjust the pH to 8,0 with concentrated HCl.
ISO 21286:2019(E)
5.4.3 Deoxynucleoside triphosphates (dNTPs).
5.4.4 PCR buffer, without Mg (500 mmol/l KCl, 100 mmol/l Tris-HCl, pH 8,3 at 25 °C).
Buffer is usually supplied with each enzyme as a 10-fold or fivefold concentrate. Use only the buffer
supplied with each particular enzyme.
5.4.5 Magnesium chloride, MgCl .
5.4.6 PCR additives (optional): trehalose dihydrate, bovine serum albumin (BSA), formamide,
dimethyl sulfoxide (DMSO).
5.4.7 Agarose (analytical grade, standard melting temperature).
5.4.8 TAE (gel-running buffer), 50-fold stock solution, pH 8,3.
Dissolve 242 g of Tris base [tris(hydroxymethyl)aminomethane], 57,1 ml of glacial acetic acid
(17,4 mol/l), 100 ml of 500 mmol/l EDTA solution (pH 8,0) in 842,9 ml of molecular grade water.
5.4.9 Size standard 100 base pair (bp) DNA ladder, a commercially available molecular-weight
marker suitable for sizing double-stranded DNA from 100 to 1 000 base pairs during gel electrophoresis.
5.4.10 6-fold Loading buffer, 3 ml of 100 % glycerol, 0,025 g of bromophenol blue, 0,025 g of xylene
cyanol FF in 7 ml of molecular grade water.
5.4.11 Ethidium bromide solution (0,5 μg/ml) or any safer alternative nucleic acid stain.
5.4.12 PCR purification kit, either using enzymatic reactions, magnetic beads or silica-membrane-
based cleanup.
5.4.13 5-fold Sequencing buffer (400 nm Tris-HCl, pH 9,0, 10 mmol/l MgCl ).
1)
5.4.14 BigDye® Terminator v3.1 cycle sequencing kit .
1)
5.4.15 Pop-7 Polymer for 3730 DNA analyzers .
1)
5.4.16 3730 DNA analyser capillary array, 50 cm .
1)
5.4.17 GeneScanTM 500 LIZTM DYE Size Standard .
5.4.18 Highly deionized formamide.
6 Apparatus
The usual laboratory equipment, including micropipettes, centrifuge, and the following specific
equipment.
6.1 Spectrophotometer, to measure the concentration and purity of double-stranded DNA at 260 nm.
1) This protocol has been validated using the 3730 DNA Analyzer capillary electrophoresis system and the
BigDye terminator chemistry. They are registered trademarks of Applied Biosystems. This information is given for
the convenience of users of this document and does not constitute an endorsement by ISO of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
4 © ISO 2019 – All rights reserved

ISO 21286:2019(E)
6.2 Laminar flow hood.
6.3 PCR thermal cycler.
6.4 Horizontal electrophoresis system.
6.5 Electrophoresis power supply.
6.6 Gel documentation system.
6.7 Automated DNA sequencing system, for DNA sequencing (e.g. 3730 DNA analyser, Applied
2)
Biosystems) .
7 General requirements
7.1 Experimental precaution and contamination avoidance
Good laboratory practice and specific anti-contamination strategies are necessary to minimize
the chance of contamination during the DNA isolation and PCR steps, either from DNA previously
handled in the laboratory, amplicon carry-over from previous PCR assays, or sample-to-sample cross-
contamination.
There are some basic steps to be followed to prevent exogenous contamination and sample carry-over:
work on a clean surface, wear gloves, and use disposable or sterilised instruments. If possible, laboratories
should use separate rooms – or, at least, different benchtops and work spaces of the laboratory – for
template extraction, PCR reagent preparation, and amplification. Work shall always flow from the
cleanest to the dirtiest area. Each work area should have dedicated supplies and reagents, as well as lab
coats and gloves. It is recommended that the setting up of PCR reactions is performed in a laminar flow
hood. It is also necessary to use sterile plastic-ware and aerosol-resistant filtered pipette tips.
When handling multiple specimens, care shall be taken to avoid cross-contamination between samples.
Anything (gloves, surfaces) that comes in contact with one sample shall be discarded or cleaned before
proceeding with the next one by wiping it with 1 % bleach solution and then thoroughly rinsing it with
water. Tools used to collect a fragment of tissue (tweezers, scissors, scalpels, etc.) shall be sterilized
before and after handling each sample. To do so, soak the instruments into ethanol (70 % to 96 %) and
then hold them briefly over the flame of a Bunsen burner or a lighter to burn off the alcohol.
7.2 Safety precautions
7.2.1 Chemical hazards
WARNING 1 — Ethidium bromide (EtBr) staining is commonly used to visualize DNA in agarose
gels. EtBr is a potential mutagen and is a skin, eye, and respiratory irritant. Avoid direct skin
contact, wear nitrile gloves, and use adequate eye protection. All staining of gels should be
done in a designated area in the laboratory, and all EtBr waste should be disposed of in labelled
containers.
2) This protocol has been validated using the 3730 DNA Analyzer capillary electrophoresis system and the
BigDye terminator chemistry. They are registered trademarks of Applied Biosystems. This information is given for
the convenience of users of this document and does not constitute an endorsement by ISO of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
ISO 21286:2019(E)
WARNING 2 — In the past few years, diverse alternatives to EtBr (e.g. GreenSafe®, Sybr® Safe,
3)
GelRed™) have become commercially available. These products are generally considered to be
less hazardous than EtBr. They are, however, still mutagenic and should also be handled and
disposed of with care.
WARNING 3 — Formamide used in sequencing reactions causes eye, skin, and respiratory tract
irritation. It is a possible developmental and birth defect hazard. When handling formamide,
wear appropriate protective eyewear, clothing, and gloves.
7.2.2 Physical hazards
Electrophoresis experiments present a potential electrical hazard if used incorrectly. Electrophoresis
units and their power supplies shall be routinely inspected to ensure that they are working properly,
that wires, leads, and connectors are undamaged and properly insulated, and that buffer tanks
have no cracks or leaks. During electrophoresis, since any wet surface can become conductive, it is
recommended not to touch any part of the equipment (tank, wires) while the power supply is on.
Ultraviolet (UV) transilluminators are often used to visualize fluorescent dyes used in gel
electrophoresis and pose potential exposures to UV radiation. Laboratory coats, gloves and appropriate
glasses and face visor shall be worn if there is any risk of exposure, especially when using unshielded
transilluminators.
8 Procedure
8.1 DNA isolation
This process can either be carried out by using a commercial DNA isolation kit or by following a
standard DNA isolation protocol. The DNA isolation kit or protocol can be selected depending on the
specific features of the sample. For instance, for fresh or recently collected tissue a Chelex-based DNA
release method usually provides enough DNA for DNA barcoding. However, for archival samples or
very small specimens, more sensitive approaches should be used (e.g. silica membrane-based DNA
[23]
extraction methods) . Using commercial kits can be helpful, as the pre-prepared reagents facilitate
the standardization of the technique.
In the case of minute organisms (i.e. length < 0,5 cm) — such as collembolans, mites, or small insects
— the entire specimen may be processed. For larger organisms, various body parts can be selected,
including muscle biopsies, abdomen, legs, antennae, or eggs. When possible, external body surfaces, the
digestive tract and body parts containing hairs, bristles, or hardened exoskeletons, should be avoided.
For earthworms, potworms, and other annelids, it is recommended to keep the specimens for about
24 h on moist filter-paper before tissue sampling, so their gut contents are evacuated.
DNA isolation from plant tissue can be challenging due to their high content in complex
polysaccharides, polyphenols, and other secondary metabolites that can affect DNA quality and inhibit
downstream reactions. Compounds such as CTAB (cetyl trimethylammonium bromide) and PVP
(polyvinylpyrrolidone) can aid in removing polysaccharides and polyphenols, respectively, and are
therefore widely used in plant DNA isolation buffers.
Detailed protocols for DNA isolation from either animal or plant tissues can be found in Reference [43]
and at the DNA barcoding website of the DNA Learning Center (http: //www .dnabarcoding101 .org/).
A negative control (with no tissue) shall be run in parallel with all batches of sample extraction, in
order to detect DNA contamination of the analytical reagents or sample-to-sample contamination. This

3) GreenSafe®, Sybr® Safe, GelRed™ are registered trademarks of NZYTech, Thermo Fisher Scientific, and Biotium,
respectively. This information is given for the convenience of users of this document and does not constitute an
endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the
same results.
6 © ISO 2019 – All rights reserved

ISO 21286:2019(E)
negative control shall be processed through the DNA isolation and PCR steps, in parallel with the test
samples.
8.2 Quantification
After DNA isolation, the yield and purity of the DNA sample should be determined by either
a) using a spectrophotometer equipped with a UV lamp, to measure sample absorbance at 260 nm, or
b) using a fluorometer that employs fluorescent DNA-binding dyes to specifically quantify double-
stranded DNA.
Typically, the DNA sample is diluted to 10 ng/µl using molecular grade water and should be stored at
−20 °C for extended periods, or at 4 °C until use.
8.3 PCR
8.3.1 Target genomic region
The target genomic region (or DNA barcode) varies across taxa. The standard DNA barcode for almost
all animal groups is a 658-base pairs region of the mitochondrial cytochrome c oxidase 1 gene (COI).
For land plants, a combination of the chloroplastic genes matK and rbcL has been recommended as the
[8]
plant DNA barcode , although the success of species discrimination using this combination can be
limited in certain plant groups.
Please note that the usual DNA barcode may not be sufficient in particular cases. In these cases,
additional genomic regions may be analysed.
8.3.2 Primer design
A critical factor in the PCR stage is the selection of appropriate primer pairs. Therefore, care shall be
taken when choosing or designing optimal primers.
The first step is to check the literature and databases (e.g. the BOLD Primer Database, available at http:
//www .boldsystems .org/index .php/Public _Primer _PrimerSearch) for existing primers that have been
successfully used to amplify the DNA barcode in the target organism group.
If there are no public primers available, or the selected primer pairs do not work on the target species,
then new primer pairs may be designed. Sequences of the same genomic region belonging to the same
or related taxa, publicly available in the BOLD database (http: //www .boldsystems .org/) and the
National Center for Biotechnology Information (NCBI) GenBank database (https: //www .ncbi .nlm .nih
[45]
.gov/genbank), can be used for primer design. Free online resources such as Primer-BLAST are
a useful tool to find primers specific to the target genomic region. This program also checks primer
specificity against a user-selected database, to confirm that the designed primers do not amplify
genomic regions other than the specific target region.
8.3.3 Primer synthesis
Primers may be ordered from a primer supplier who specializes in the synthesis of oligonucleotides. A
standard desalting purification is sufficient for the primers used in DNA barcoding.
The primers are usually shipped and delivered in a lyophilized state in nanomoles quantity. Before
PCR, they should be centrifuged and resuspended in TE buffer or molecular grade water to make a
100 µmol/l stock. Alternatively, primers can be ordered already resuspended at a given concentration
(typically between 10 µmol/l to 100 µmol/l) from several manufacturers. In either case, resuspended
primers shall be dispensed into single-use aliquots and stored at −20 °C.
ISO 21286:2019(E)
8.3.4 PCR
For standard PCR, reactions are set up in sterile, thin-walled 0,2 ml tubes or an equivalent PCR plate
format. The final concentrations of reagents for a typical PCR reaction, with a final volume of 25 µl, are
as follows (see Table 1).
Table 1 — Final concentrations of reagents for a typical PCR reaction, with a final volume of 25 µl
Volume
Reagent Initial concentration Final concentration
µl
PCR buffer 10-fold 2,5 1-fold
MgCl 50 mmol/l 0,75 1,5 mmol/l
dNTPs 2,5 mmol/l 2,0 0,2 mmol/l
Forward primer 0,1 mmol/l 0,125 0,5 µmol/l
Reverse primer 0,1 mmol/l 0,125 0,5 µmol/l
Taq polymerase 5 units/µl 0,125 0,025 units/µl
Template DNA 10 ng/µl 2,5 1 ng/µl
Nuclease-free sterile water 16,875
Always add water and buffer first and Taq polymerase last.
When multiple PCR reactions are set up simultaneously, it is useful to prepare a master mix that
contains all the reagents except for DNA template. This reduces preparation time and the potential for
pipetting errors. Make up enough master mix for the number of reactions to be amplified, and an extra
volume to allow for pipetting errors (as a rule of thumb, count one extra reaction every 10 samples).
Addition of PCR-enhancing agents in the PCR mix can increase the yield of the specific PCR product and
overcome the effect on PCR inhibitors present in the reaction. Some of these enhancers are trehalose,
bovine serum albumin (BSA), betaine, formamide or DMSO (e.g. References [18] and [39]). Alternatively,
pre-optimised commercial master mixes may be used.
The high sensitivity of PCR makes it especially vulnerable to trace contamination. Therefore, it is
necessary to use sterile tips (and preferably filter tips) for pipetting the PCR reagents. DNA templates
(and other PCR products) shall also be kept away from the PCR reagents while setting up the PCR
reaction mix. In addition, and besides the DNA isolation negative control, every set of reactions shall
include a PCR negative control, which contains all reagents except for the DNA template. This PCR
negative control is used to detect DNA contamination in the PCR reagents.
PCR is performed in a thermocycler (PCR block), according to the following program:
— one cycle of 4 min at 94 °C;
— 35 cycles of: 30 s at 94 °C, 40 s at specific annealing temperature (Ta) for the PCR primers, 1 min
at 72 °C;
— a final extension step
...