Soil quality - Direct extraction of soil DNA (ISO 11063:2020)

The present document specifies a method for direct extraction of DNA from soil samples to analyse the abundance and composition of microbial communities by various techniques of molecular biology including real-time quantitative PCR (qPCR). This method is mainly dedicated to agricultural and forest soils. This method can possibly not be suitable for soils rich in organic matter (e.g. peat soils) or soils heavily polluted with organic pollutants or heavy metals.
The direct extraction of DNA from soil samples provides unique insight into the α- and β-diversity of microbial communities. Next-generation sequencing of amplicons obtained by PCR (polymerase chain reaction) amplification of soil DNA constitutes a promising domain which will in the near future contribute to the development of routine tools to monitor microbial communities in soil environments.

Bodenbeschaffenheit - Direkte Extraktion von DNA aus Bodenproben (ISO 11063:2020)

Dieses Dokument legt ein Verfahren zur direkten Extraktion von DNA aus Bodenproben für die Analyse der Abundanz und Zusammensetzung von mikrobiellen Lebensgemeinschaften mit verschiedenen Methoden der Molekularbiologie einschließlich quantitative Echtzeit PCR (real-time qPCR) fest. Dieses Verfahren ist vorwiegend für landwirtschaftlich genutzte Böden und Waldboden anzuwenden. Es ist möglicherweise nicht anzuwenden für Böden mit hohem organischen Anteil (z. B. Torfböden) oder Böden, die stark mit organischen Verunreinigungen oder Schwermetallen belastet sind.
Die direkte Extraktion von DNA aus Bodenproben bietet einen einzigartigen Einblick in die α  und β Diversität von mikrobiellen Lebensgemeinschaften. Next-Generation Sequencing von Amplikons, die durch PCR Amplifikation von Boden-DNA erhalten wurden, stellen einen vielversprechenden Ansatz dar und können in naher Zukunft zur Entwicklung von Routinewerkzeugen beitragen, die die Abschätzung der mikrobiellen Gemeinschaften im Boden ermöglichen.

Qualité du sol - Extraction directe de l'ADN du sol (ISO 11063:2020)

Le présent document spécifie une méthode pour l'extraction directe de l'ADN des échantillons de sol afin d'analyser l'abondance et la composition des communautés microbiennes au moyen de différentes techniques de biologie moléculaire, y compris la PCR quantitative en temps réel (qPCR). Cette méthode est principalement destinée aux sols agricoles et forestiers. Cette méthode peut ne pas être appropriée aux sols riches en matières organiques (par exemple sols de tourbières) ou aux sols très pollués par des polluants organiques ou des métaux lourds.
L'extraction directe de l'ADN des échantillons de sol fournit des informations précieuses sur la diversité α et β des communautés microbiennes. Le séquençage de dernière génération des amplicons obtenus par amplification PCR (amplification en chaîne par polymérase) de l'ADN extrait du sol offre des perspectives prometteuses et pourra contribuer, dans un avenir proche, au développement d'outils de routine permettant de surveiller les communautés microbiennes des sols.

Kakovost tal - Neposredna ekstrakcija DNK iz vzorcev tal (ISO 11063:2020)

General Information

Status
Published
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
14-Oct-2020
Completion Date
14-Oct-2020

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SLOVENSKI STANDARD
SIST EN ISO 11063:2020
01-december-2020
Nadomešča:
SIST EN ISO 11063:2013
Kakovost tal - Neposredna ekstrakcija DNK iz vzorcev tal (ISO 11063:2020)
Soil quality - Direct extraction of soil DNA (ISO 11063:2020)

Bodenbeschaffenheit - Direkte Extraktion von DNA aus Bodenproben (ISO 11063:2020)

Qualité du sol - Extraction directe de l'ADN du sol (ISO 11063:2020)
Ta slovenski standard je istoveten z: EN ISO 11063:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
SIST EN ISO 11063:2020 en,fr,de

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

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SIST EN ISO 11063:2020
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SIST EN ISO 11063:2020
EN ISO 11063
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2020
EUROPÄISCHE NORM
ICS 13.080.30 Supersedes EN ISO 11063:2013
English Version
Soil quality - Direct extraction of soil DNA (ISO
11063:2020)

Qualité du sol - Extraction directe de l'ADN du sol (ISO Bodenbeschaffenheit - Direkte Extraktion von DNA aus

11063:2020) Bodenproben (ISO 11063:2020)
This European Standard was approved by CEN on 18 September 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 11063:2020 E

worldwide for CEN national Members.
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SIST EN ISO 11063:2020
EN ISO 11063:2020 (E)
Contents Page

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

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SIST EN ISO 11063:2020
EN ISO 11063:2020 (E)
European foreword

This document (EN ISO 11063:2020) has been prepared by Technical Committee ISO/TC 190 "Soil

quality" in collaboration with 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 April 2021, and conflicting national standards shall be

withdrawn at the latest by April 2021.

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.

This document supersedes EN ISO 11063:2013.

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 11063:2020 has been approved by CEN as EN ISO 11063:2020 without any modification.

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SIST EN ISO 11063:2020
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SIST EN ISO 11063:2020
INTERNATIONAL ISO
STANDARD 11063
Second edition
2020-09
Soil quality — Direct extraction of
soil DNA
Qualité du sol — Extraction directe de l'ADN du sol
Reference number
ISO 11063:2020(E)
ISO 2020
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SIST EN ISO 11063:2020
ISO 11063:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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SIST EN ISO 11063:2020
ISO 11063:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 1

5 Test materials .......................................................................................................................................................................................................... 2

5.1 Soil ..................................................................................................................................................................................................................... 2

5.2 Chemicals ..................................................................................................................................................................................................... 2

5.3 Buffers and reagents .......................................................................................................................................................................... 3

6 Apparatus ..................................................................................................................................................................................................................... 4

7 Procedures .................................................................................................................................................................................................................. 4

7.1 Preparation of soil samples .......................................................................................................................................................... 4

7.2 Mechanical and chemical lyses ................................................................................................................................................. 4

7.3 Protein precipitation .......................................................................................................................................................................... 4

7.4 Nucleic acid precipitation and washing ............................................................................................................................ 4

7.5 Nucleic acid storage ............................................................................................................................................................................ 5

8 Estimation of soil DNA quality and quantity ........................................................................................................................... 5

8.1 Soil DNA quality and purity ......................................................................................................................................................... 5

8.2 Soil DNA quantity .................................................................................................................................................................................. 5

9 Validation of the extraction procedure ......................................................................................................................................... 5

10 Test report ................................................................................................................................................................................................................... 6

Annex A (informative) Differences between ISO 11063:2012 and the revised document for

direct extraction of DNA from soil samples .............................................................................................................................. 7

Annex B (informative) Possible methods to purify soil DNA extracts ................................................................................ 8

Bibliography ................................................................................................................................................................................................................................ 9

© ISO 2020 – All rights reserved iii
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SIST EN ISO 11063:2020
ISO 11063:2020(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/SC 4, Soil quality, Subcommittee SC 4

Biological characterization

This second edition cancels and replaces the first edition (ISO 11063:2012), which has been technically

revised. The main changes compared to the previous edition are as follows (see details in Annex A):

— the amount of soil used (1 g instead of 0,25 g dry mass equivalent);

— the nature and amount of beads (2,5 g of ceramic beads, 2 g of 0,1 mm silica beads and 4 glass beads

instead of 0,5 g of 106 µm glass beads and 2 glass beads);
— the duration of the mechanical lysis (90 s instead of 30 s);

— the salt used to precipitate the proteins (potassium acetate instead of sodium acetate).

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 2020 – All rights reserved
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SIST EN ISO 11063:2020
ISO 11063:2020(E)
Introduction

DNA (deoxyribonucleic acid) is an essential component of any living organism coding for enzymes

responsible for any biological activities. The study of DNA sequences from DNA sources extracted from

different matrices, by means of numerous molecular approaches, provides molecular markers that can

be used to sharply distinguish and identify different organisms (bacteria, archaea and eucaryotes).

Up to now, most of the studies aiming to develop microbial soil quality indicators applicable to complex

environments, such as soil, were biased by the unculturability of many microorganisms and the lack of

[1]

sensitivity of traditional microbiological methods. The recent development of numerous molecular

biology methods based primarily on amplification of soil-extracted nucleic acids have provided a

pertinent alternative to classical culture-based microbiological methods, providing unique insight into

[2],[3],[4],[5],[6]

the composition, richness, and structure of microbial communities. DNA-based approaches

are now well-established in soil ecology and serve as genotypic (molecular genetic) markers for

determining microbial diversity.

The results of molecular analyses of soil microbial communities and/or populations rely on two main

parameters:

a) the extraction of DNA representative of the indigenous bacterial community composition;

b) PCR bias, such as the choice of primers, the concentration of amplified DNA, errors in the PCR, or

[4],[7],[8],[9]

even the method chosen for analysis. Recently, numerous studies have investigated new

[10]

methods to improve extraction, purification, amplification, and quantification of DNA from soils .

The first edition (ISO 11063:2012) described the procedure used to extract DNA directly from soil

samples suitable for the study of the composition of microbial community by both quantitative (q-PCR)

[11]
and qualitative (A-RISA) approaches .
[12]

The aim of this document is to describe a new method recently reported derived from the first

edition procedure to analyse the diversity of soil microorganisms by next-generation sequencing of

ribosomal amplicons generated by polymerase chain reactions (PCR) using soil DNA as template. This

new method was shown to increase the DNA recovery and to better represents the abundance and the

[12]

structure of archaeal and fungal communities as compared to the original method .

© ISO 2020 – All rights reserved v
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SIST EN ISO 11063:2020
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SIST EN ISO 11063:2020
INTERNATIONAL STANDARD ISO 11063:2020(E)
Soil quality — Direct extraction of soil DNA
1 Scope

The present document specifies a method for direct extraction of DNA from soil samples to analyse

the abundance and composition of microbial communities by various techniques of molecular biology

including real-time quantitative PCR (qPCR). This method is mainly dedicated to agricultural and forest

soils. This method can possibly not be suitable for soils rich in organic matter (e.g. peat soils) or soils

heavily polluted with organic pollutants or heavy metals.

The direct extraction of DNA from soil samples provides unique insight into the α- and β-diversity

of microbial communities. Next-generation sequencing of amplicons obtained by PCR (polymerase

chain reaction) amplification of soil DNA constitutes a promising domain which will in the near future

contribute to the development of routine tools to monitor microbial communities in soil environments.

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 18400-206, Soil quality — Sampling — Part 206: Collection, handling and storage of soil under aerobic

conditions for the assessment of microbiological processes, biomass and diversity in the laboratory

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: avai
...

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