EN ISO 11063:2013
(Main)Soil quality - Method to directly extract DNA from soil samples (ISO 11063:2012)
Soil quality - Method to directly extract DNA from soil samples (ISO 11063:2012)
This International Standard specifies a method for direct extraction of DNA from soil samples to analyse the
global structure and the abundance of soil bacterial communities using PCR-based technologies. 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 richness and structure of
microbial communities which are key parameters to estimate the biodiversity of soil microbiota. Molecular
approaches based on PCR (polymerase chain reaction) amplification of soil DNA constitute a promising
domain and can contribute in the near future to the development of routine tools to monitor the microbiota of
soil environments.
Users of the method ought to be aware that although soil submitted to the DNA extraction procedure is sieved
thoroughly (2 mm mesh, procedure described in 5.1), plant residues can still remain in soil samples and, as a
result, traces of plant DNA can contaminate the soil DNA extract.
Bodenbeschaffenheit - Verfahren zur direkten Extraktion von DNA aus Bodenproben (ISO 11063:2012)
Diese Norm legt ein Verfahren zur direkten Extraktion von DNA aus Bodenproben für die Analyse der allgemeinen Struktur und der Abundanz von Lebensgemeinschaften von Bodenbakterien mit Verfahrensweisen auf der Grundlage von PCR fest. Dieses Verfahren ist vorwiegend für landwirtschaftlich genutzte Böden und Waldboden vorgesehen. Dieses Verfahren kann möglicherweise für Böden mit hohem organischen Anteil (z. B. Torfböden) oder Böden, die stark mit organischen Verunreinigungen oder Schwermetallen belastet sind, geeignet sein.
Die direkte Extraktion von DNA aus Bodenproben bietet einen einzigartigen Einblick in den Artenreichtum und die Struktur mikrobieller Lebensgemeinschaften, was die Schlüsselparameter zur Abschätzung der Biodiver-sität der Bodenmikroflora und -fauna sind. Molekulare Verfahren auf der Grundlage der Amplifikation von Boden DNA durch PCR (Polymerasekettenreaktion) stellen ein vielversprechendes Gebiet dar und können in naher Zukunft zur Entwicklung von Routinewerkzeugen beitragen, die die Abschätzung der Bodenbeschaf-fenheit ermöglichen.
Anwender dieser Norm sollten beachten, dass obwohl der Boden für die DNA-Extraktion sorgfältig gesiebt wird (2-mm-Sieb, Verfahren in 5.1 beschrieben), Pflanzenrückstände in den Bodenproben noch verbleiben können und damit Spuren von Pflanzen-DNA das Boden-Extrakt verunreinigen können.
Qualité du sol - Méthode pour extraire directement l'ADN d'échantillons de sol (ISO 11063:2012)
La présente Norme internationale spécifie une méthode pour extraire directement l'ADN d'échantillons de
sol en vue d'analyser la structure globale et l'abondance des communautés microbiennes du sol en utilisant
des techniques de PCR. 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 d'échantillons de sol fournit des informations précieuses sur l'abondance et
la structure des communautés microbiennes qui sont des paramètres clés pour estimer la biodiversité des
communautés microbiennes telluriques. Les approches moléculaires utilisant l'amplification PCR (amplification
en chaîne par polymérisation) de l'ADN extrait du sol offrent des perspectives prometteuses et peuvent
contribuer, dans un futur proche, au développement d'outils de routine permettant de surveiller la qualité de la
composante microbienne des sols.
L'utilisateur de la méthode doit savoir que, même si le sol soumis au mode opératoire d'extraction de l'ADN
est soigneusement tamisé (mailles de 2 mm, mode opératoire décrit en 5.1), des résidus végétaux peuvent
demeurer dans les échantillons de sol et de ce fait, l'extrait d'ADN prélevé du sol peut être contaminé par des
traces d'ADN végétal.
Kakovost tal - Metoda neposredne ekstrakcije DNK iz vzorcev tal (ISO 11063:2012)
Ta mednarodni standard določa metodo neposredne ekstrakcije DNK iz vzorcev tal za analizo globalne strukture in številčnosti bakterijskih skupnosti v tleh z uporabo tehnologij na podlagi polimerazne verižne reakcije (PCR). Ta metoda se uporablja predvsem za kmetijska in gozdna tla. Ta metoda je lahko neprimerna za tla, bogata z organsko snovjo (npr. šotna tla), ali tla, ki so močno onesnažena z organskimi onesnaževali ali težkimi kovinami. Neposredna ekstrakcija DNK iz vzorcev tal omogoča enkraten vpogled v bogatost in strukturo mikrobnih skupnosti, ki so ključni parametri za oceno biotske raznovrstnosti talne mikrobiote. Molekularni pristopi, ki temeljijo na okrepitvi PCR (polimerazne verižne reakcije) DNK v tleh, predstavljajo obetavno domeno in lahko v bližnji prihodnosti prispevajo k razvoju rutinskih orodij za spremljanje mikrobiote talnih okolij. Uporabniki metode se morajo zavedati, da lahko kljub temeljitemu presejanju tal v postopku ekstrakcije DNK (z 2 mm mrežo, postopek je opisan v točki 5.1), v vzorcih tal še vedno ostanejo ostanki rastlin, zato lahko sledovi rastlinskega DNK onesnažijo ekstrakt DNK tal.
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
01-maj-2013
Kakovost tal - Metoda neposredne ekstrakcije DNK iz vzorcev tal (ISO 11063:2012)
Soil quality - Method to directly extract DNA from soil samples (ISO 11063:2012)
Bodenbeschaffenheit - Verfahren zur direkten Extraktion von DNA aus Bodenproben
(ISO 11063:2012)
Qualité du sol - Méthode pour extraire directement l'ADN d'échantillons de sol (ISO
11063:2012)
Ta slovenski standard je istoveten z: EN ISO 11063:2013
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.
EUROPEAN STANDARD
EN ISO 11063
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2013
ICS 13.080.30
English Version
Soil quality - Method to directly extract DNA from soil samples
(ISO 11063:2012)
Qualité du sol - Méthode pour extraire directement l'ADN Bodenbeschaffenheit - Verfahren zur direkten Extraktion
d'échantillons de sol (ISO 11063:2012) von DNA aus Bodenproben (ISO 11063:2012)
This European Standard was approved by CEN on 5 February 2013.
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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11063:2013: E
worldwide for CEN national Members.
Contents Page
Foreword . 3
Foreword
The text of ISO 11063:2012 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 11063:2013 by
Technical Committee CEN/TC 345 “Characterization of soils” 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 August 2013, and conflicting national standards shall be withdrawn at
the latest by August 2013.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 11063:2012 has been approved by CEN as EN ISO 11063:2013 without any modification.
INTERNATIONAL ISO
STANDARD 11063
First edition
2012-02-01
Soil quality — Method to directly extract
DNA from soil samples
Qualité du sol — Méthode pour extraire directement l’ADN
d’échantillons de sol
Reference number
ISO 11063:2012(E)
©
ISO 2012
ISO 11063:2012(E)
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
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ii © ISO 2012 – All rights reserved
ISO 11063:2012(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 . 4
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 International ring test . 5
11 Test report . 5
Annex A (informative) International ring test for evaluating soil DNA extraction procedure . 7
Bibliography .21
ISO 11063:2012(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 11063 was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.
iv © ISO 2012 – All rights reserved
ISO 11063:2012(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 matrixes,
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 sensitivity
[16]
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 the composition, richness, and structure
[15], [18], [26], [27], [36]
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
[23], [26], [38], [40]
even the method chosen for analysis . Recently, numerous studies have investigated new
[20]
methods to improve extraction, purification, amplification, and quantification of DNA from soils .
The aim of this International Standard is to describe the procedure used to extract DNA directly from soil
samples. The reproducibility of this soil DNA extraction procedure was assessed in an international ring-test
study (Annex A). The reproducibility of this soil DNA extraction procedure was successfully evaluated on both
quantitative (q-PCR) and qualitative (A-RISA) approaches.
INTERNATIONAL STANDARD ISO 11063:2012(E)
Soil quality — Method to directly extract DNA from soil samples
1 Scope
This International Standard specifies a method for direct extraction of DNA from soil samples to analyse the
global structure and the abundance of soil bacterial communities using PCR-based technologies. 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 richness and structure of
microbial communities which are key parameters to estimate the biodiversity of soil microbiota. Molecular
approaches based on PCR (polymerase chain reaction) amplification of soil DNA constitute a promising
domain and can contribute in the near future to the development of routine tools to monitor the microbiota of
soil environments.
Users of the method ought to be aware that although soil submitted to the DNA extraction procedure is sieved
thoroughly (2 mm mesh, procedure described in 5.1), plant residues ca
...
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