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
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
17-Aug-2020
Due Date
22-Oct-2020
Completion Date
24-Sep-2020

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SLOVENSKI STANDARD
SIST EN ISO 21286:2020
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
SIST EN ISO 21286:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 21286:2020
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SIST EN ISO 21286:2020
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.
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SIST EN ISO 21286:2020
EN ISO 21286:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 21286:2020
EN ISO 21286:2020 (E)
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.

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SIST EN ISO 21286:2020
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SIST EN ISO 21286:2020
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
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SIST EN ISO 21286:2020
ISO 21286:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 21286:2020
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 2019 – All rights reserved iii
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SIST EN ISO 21286:2020
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
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SIST EN ISO 21286:2020
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 .
© ISO 2019 – All rights reserved v
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SIST EN ISO 21286:2020
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SIST EN ISO 21286:2020
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 2019 – All rights reserved 1
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SIST EN ISO 21286:2020
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
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SIST EN ISO 21286:2020
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 2019 – All rights reserved 3
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SIST EN ISO 21
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