EN ISO 11063:2013

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
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
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Published in Switzerland
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 can still remain in soil samples and, as a
result, traces of plant DNA can contaminate the soil DNA extract.
2 Normative references
The following referenced documents are indispensable for the application 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 10381-6, Soil quality — Sampling — Part 6: Guidance on the 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.
3.1
soil DNA
DNA extracted from soil-living microorganisms and remaining DNA from dead microorganisms
4 Principle
DNA is directly extracted from 0,25 g soil samples using the following extraction procedure. This method
reliably allowed analysing the global structure of bacterial and archeal communities and could be adapted
[32]
(extraction from a 1 g soil sample) to assess the global structure of fungal communities . Soil samples added
with an extraction buffer are submitted to mechanical and chemical lyses. The lysis step, e.g. by bead beating,
is a crucial step to also extract DNA from microbes that are difficult to lyse. After a brief centrifugation, soil
debris are removed and proteins are precipitated with potassium acetate. After centrifugation, the supernatant
is recovered and nucleic acids are precipitated with ice-cold isopropanol. After centrifugation, the nucleic acids
pellet is washed with 70 % ethanol and suspended in sterile ultra-pure water. DNA quality is then checked by
electrophoresis on an agarose gel and the DNA quantity is estimated using a spectro-fluorimeter. A schematic
overview of the procedure is given in Figure 1.
ISO 11063:2012(E)
5 Test materials
5.1 Soil
Soil samples should be collected and sieved (2 mm mesh). If samples are not immediately processed, they
should be stored for up to two years at −20 °C or up to 10 years at −80 °C or in liquid nitrogen (−180 °C) as
specified in ISO 10381-6. If soil samples are frozen, they may be thawed only once. Some of these storage
conditions are currently under testing.
Figure 1 — Schematic overview of soil DNA extraction procedure
5.2 Chemicals
5.2.1 Tris[hydroxymethyl]aminomethane, C H NO (CAS No. 77-86-1).
4 11 3
5.2.2 Ethylenediaminetetraacetic acid disodium salt (EDTA), C H N O Na ·2 H O (CAS No. 6381-92 6).
10 14 2 8 2 2
5.2.3 Sodium chloride, NaCl (CAS No. 7647-14-5).
2 © ISO 2012 – All rights reserved

ISO 11063:2012(E)
5.2.4 Sodium dodecyl sulfate (SDS), CH (CH ) OSO Na (CAS No. 151-21-3).
3 2 11 3
5.2.5 Polyvinylpyrrolidone (PVP), [C H NO] (CAS No. 9003-39-8).
6 9 n
5.2.6 Sodium acetate, CH COONa (CAS No. 6131-90-4).
5.2.7 Acetic acid or glacial acetic acid, CH COOH (CAS No. 64-19-7).
5.2.8. Isopropanol, CH CHOHCH (CAS No. 67-63-0).
3 3
5.2.9 Ethanol, CH CH OH (CAS No. 64-17-5).
3 2
5.2.10 Molecular-biology-grade water, H O.
5.3 Buffers and reagents
Buffers and reagents (except intercalent molecules) used for soil DNA extraction are sterilized (120 °C for
20 min) and stored at room temperature. Ethanol and isopropanol are stored at −20 °C.
5.3.1 Tris-HCl, 1 mol/l, 121,14 g of tris in 1 000 ml of H O, adjusting with 4 mol/l HCl to pH 8,0.
5.3.2 EDTA, 0,5 mol/l, 186,10 g of EDTA in 1 000 ml of H O, adjusting with NaOH (10 mol/l) to pH 8,0.
5.3.3 NaCl, 1 mol/l, 58,44 g of NaCl in 1 000 ml of H O.
5.3.4 PVP 40, 20 %, 200 g of PVP in 1 000 ml of H O.
5.3.5 SDS, 20 %, 200 g of SDS in 1 000 ml of H O.
5.3.6 Homogenization buffer (newly prepared just before being used), 100 ml of 1 mol/l tris-HCl (pH 8,0), 200 ml
of 0,5 mol/l EDTA (pH 8,0), 100 ml of 1 mol/l NaCl, 50 ml of 20 % PVP 40, 100 ml of 20 % SDS in 450 ml of H O.
5.3.7 Sodium acetate, 5 mol/l (pH 5,5), 410,15 g of CH COONa in 800 ml of H O. Add 120 ml of acetic acid
3 2
and then adjust the pH to 5,5 with glacial acetic acid. Add water to make up to 1 000 ml.
5.3.8 Ethanol, 70 %, 700 ml of pure ethanol in 300 ml of H O.
5.3.9 TE buffer, pH 8,0, 10 mmol/l tris-HCl, 1 mmol/l EDTA.
5.3.10 Glass beads (106 µm).
5.3.11 Glass beads (2 mm).
5.3.12 Ethidium bromide, 5 mg of ethidium bromide in 1 000 ml of H O.
5.3.13 Fluorescent nucleic acid stain, excitation at 480 nm and emission at 520 nm.
5.3.14 Pure DNA (100 ng/µl)
5.3.15 TBE buffer × 10, pH 8,0, 108 g of tris base, 55 g of boric acid, 40 ml of 0,5 mol/l EDTA (pH 8,0) in
1 000 ml of H O.
ISO 11063:2012(E)
5.3.16 TBE buffer × 1, 100 ml of TBE buffer × 10 in 900 ml of H O.
6 Apparatus
Use standard laboratory equipment including pipettes, a centrifuge, fume hood cabinet, horizontal
electrophoresis system and the following.
-1
6.1 Mini-bead beating apparatus, with a beating frequency varying from, for example, 100 min to
-1
2 600 min and a 16 mm amplitude of agitation.
6.2 Spectro-fluorimeter, allowing the quantification of double-strand DNA at 520 nm with a fluorescent
nucleic acid stain excited at 480 nm.
7 Procedures
7.1 Preparation of soil samples
Weigh 0,25 g of soil (equivalent dry mass) in 2 ml micro-tubes just before extracting, or immediately freeze the
soil sample in liquid nitrogen and keep it frozen at −80 °C until its use.
7.2 Mechanical and chemical lyses
Add 0,5 g of 106 µm glass beads (wear a mask for protection) and two glass beads (2 mm diameter) to the soil
sample. Add 1 ml of homogenization buffer (composition given in 5.3.6). Agitate the soil samples 1 600g for
30 s (16 mm of amplitude) using a bead-beating system (tube support previously placed at −20 °C). Incubate
at 70 °C for 10 min. Centrifuge for 1 min at 14 000g (4 °C). Carefully recover the supernatant and transfer it to
a new 2 ml microtube.
7.3 Protein precipitation
To the supernatant obtained in 7.2, add 5 mol/l sodium acetate (pH 5,5) (composition given in 5.3.7) of an
amount that is 1/10 of the volume of the supernatant. Mix by vortexing and incubate on ice for 10 min. Centrifuge
for 5 min at 14 000g (4 °C). Carefully recover the supernatant and transfer it to a new 1,5 ml microtube.
7.4 Nucleic acid precipitation and washing
Perform all these steps below a fume hood because of dangerous isopropanol vapours. Liquid and solid
wastes shall be evacuated as chemical waste.
To the supernatant obtained in 7.3, add cold isopropanol (-20 °C) of an amount that is 1/1 of the volume of the
supernatant. Incubate the samples at −20 °C for 15 min. Centrifuge for 30 min at 14 000g (4 °C). Carefully
eliminate the supernatant. Wash the nucleic acids pellet with cold 70 % ethanol (do not resuspend the pellet).
Centrifuge for 15 min at 14 000g (4 °C). Eliminate any traces of ethanol and let the nucleic acid pellet dry for
15 min at 37 °C. Suspend the pellet in 100 µl of ultra-pure water or TE buffer (pH 8) (composition given in 5.3.9).
7.5 Nucleic acid storage
Aliquot the soil DNA (4 × 25 µl) and store the DNA samples at −20 °C until their use. Repeated freezing and
thawing of the DNA extracts should be omitted.
4 © ISO 2012 – All rights reserved

ISO 11063:2012(E)
8 Estimation of soil DNA quality and quantity
8.1 Soil DNA quality and purity
The quality and the size of the soil DNA are checked by electrophoresis on 1 % agarose gels in TBE buffer. Gels
are stained with appropriate staining (e.g. ethidium bromide, 5 mg/l). The purity of the soil DNA is assessed by
[11]
spectrophotometry at 260 nm for the DNA analysis and at 400 nm for humic acid substances .
The step of chemical and mechanical lysis is critical, and it should be adequate to lyse a representative portion
[39]
of microbes but avoid fragmentation of the DNA .
DNA extracts which are still slightly brownish need a further DNA purification.
8.2 Soil DNA quantity
The soil DNA content is determined using a fluorescent nucleic acid stain (5.3.13) which fluoresces when
intercalated within the double helix of DNA. A calibration curve relating the amount of standard DNA (5 ng,
10 ng, 20 ng, 50 ng, 100 ng, 150 ng and 200 ng of pure DNA) to the amount of fluorescence quantified is
established and used to estimate the amount of DNA extracted from the soil. Measurements are performed
using a spectro-fluorimeter (6.2). The analysis is carried out by relevant software.
Alternatively, the soil DNA content can be determined by resolving soil DNA extracts by electrophoresis in a
1 % agarose gel, stained with ethidium bromide and photographed under a camera. Dilutions of pure DNA
were included in each gel and a standard curve of DNA concentration (1 000 ng, 500 ng, 250 ng, 125 ng,
62,5 ng to 31,25 ng). The ethidium bromide intensity was integrated to establish a standard curve used for
estimating soil DNA concentration as described previously by Reference [32].
Alternatively, the soil DNA content can be determined by spectrophotometry at 260 nm when soil DNA is lowly
contaminated with humic acid substances (400 nm) and proteins (A260/A280 averaging 1,6).
9 Validation of the extraction procedure
The laboratory can validate the procedure of soil DNA extraction by processing the reference soil and comparing
the obtained yield of soil DNA extraction to the expected one.
10 International ring test
This method for extracting soil DNA was evaluated through an international ring test involving nine different
laboratories working on six different European soils. The report of this ring test is provided in Annex A.
11 Test report
The test report shall include the following information:
a) a reference to this International Standard: ISO 11063:2012;
b) soil collection, including date and place (GPS coordinates) of collection;
c) treatment and storage of soil sample (e.g. sieving method, conditions and length of storage);
d) physical and chemical characteristics of the soil;
e) quantity of soil used for DNA extraction;
f) date(s) of extraction;
g) duration of nucleic acids storage (if appropriate);
ISO 11063:2012(E)
h) tables of results including concentration of soil DNA extracts and amount of DNA extracted per gram of
soil (dry weight equivalent);
i) any details not specified in this International Standard or which are optional, as well as any effect which
may have affected the results.
6 © ISO 2012 – All rights reserved

ISO 11063:2012(E)
Annex A
(informative)
International ring test for evaluating soil DNA extraction procedure
A.1 Introduction
Up to now, most of the microbial diversity studies conducted in complex ecosystems, such as soil, has been
biased essentially by the unculturability of many microorganisms and the lack of sensitivity of traditional
microbiological methods. In the past decade, applications of numerous molecular biology methods based
primarily on amplification of indirect or direct soil-extracted nucleic acids (DNA or DNA/RNA) have provided
a pertinent alternative to classical culture-based microbiological methods, providing unique insight into the
[15], [18], [26], [27], [36]
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.
However, the results of molecular analysis of microbial communities rely mostly on two main parameters:
a) the extraction of DNAs 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 even
the method chosen for analysis. Recently, numerous studies have investigated new methods to improve
extraction, purification, amplification, and quantification of DNA from soils.
Although comparative studies have been performed to analyse the efficiency of methods for extraction and
purification of soil DNA recovered, the reproducibility of soil DNA extraction within different laboratories has not
yet been assessed. Therefore, in this context, the aim of this study was to evaluate the reproducibility of a soil
DNA extraction method developed by Reference [20].
A.2 Materials and methods
A.2.1 Laboratories involved in the study
Nine different laboratories from six European countries (France, Finland, Germany, Spain, Italy and Sweden)
were involved in the international ring test with the following organization. Laboratory in charge of the program:
INRA/Université de Bourgogne, Laboratoire de Microbiologie du Sol et de l’Environnement, (Dijon, France).
The participant laboratories are listed below: Institut National de l’Environnement Industriel et des Risques,
INERIS (Verneuil-en-Halatte, France); IPL santé, environnement durables Est, Laboratoire Etudes et Expertises
(Nancy, France); Swedish University of Agricultural Sciences (Uppsala, Sweden); GSF Munich (Munich,
Germany); Julius Kühn-Institut Bundesforschungsinsinstitut für Kulturpflanzen, JKI (Brauschweig, Germany);
Universita di Catania, DACPA-Sezione Scienze Agrochimiche (Catania, Italy); CSIC, Estacion Experimental
del Zaidin (Grenada, Spain); University of Helsinki (Helsinki, Finland).
A working group constituted of each responsible scientist of each research laboratory was established. The
main objectives of the working group were to:
— design the set-up of the inter-laboratories assay,
— analyse and discuss the results produced in this study.
The procedure used for the international ring test study is described below. The working group decided that
this assay should only concern the evaluation of the reproducibility of soil DNA extraction. To do so, the
working group decided that each laboratory should extract DNA from different soil samples following the same
protocol. Further work (DNA purification and DNA analyses) was only done by the laboratory leading this
ISO 11063:2012(E)
project (Laboratoire de Microbiologie du Sol et de l’Environnement, Dijon, France) in order to avoid adding
several biases due to these two additional steps.
Therefore, after soil DNA extraction, each laboratory involved in this project sent their samples to “Laboratoire
de Microbiologie du Sol et de l’Environnement” for further analyses. Upon their arrival, soil DNA samples were
purified as described in A.2.4. Soil DNA extracts were quantified on agarose gel stained with ethidium bromide.
Following purification, extracted soil DNA was further analysed using PCR:
a) 16S rDNA quantitative PCR assay for estimating the total bacterial community abundance in studied soils;
b) pcaH and narG quantitative PCR assay for estimating the narG and pcaH microbial community abundances
in studied soils;
c) A-RISA method (automatic ribosomal intergenic spacer analysis) for studying the structure of the global
bacterial community.
A.2.2 Soil physico-chemical properties and preparation
The working group chose six different soils named soil A, B, C, F, M and soil S (see Table A.1) collected in
different European countries. One soil was PAH-contaminated (collected in Finland), four were agricultural
soils (collected in Sweden and France) whilst one was a forest soil (collected in Germany).
Fresh soil samples were sieved (2 mm mesh) and 250 mg aliquots were prepared in 2 ml tubes. These aliquots
were immediately frozen in liquid nitrogen and stored at −80 °C until their shipment. Five aliquots of each soil were
sent in dry ice to each laboratory where they were stored at −80 °C until soil DNA extraction were carried out.
Table A.1 — Physico-chemical characteristics of the studied soils
Soil
A B C F M S
Forest soil Agricultural Agricultural PAH- Agricultural Agricultural
soil soil contaminated soil soil
soil
Clay % 17,6 nd 43,2 15,5 43,2 47,8
Silt % 26,9 nd 50,3 26,2 23,7 26,8
Sand % 55,5 nd 6,5 58,3 33,1 25,4
Organic C % 7,55 1,45 1,29 1,12 3,27 1,53
Total N % 0,46 0,12 0,14 0,33 0,36 0,16
C/N 16,5 11,7 9,21 33,5 9,11 9,7
Organic matter % 13,1 2,51 3,25 19,3 5,65 2,66
pH 3.76 6,41 7,50 6,22 7,88 7,99
CEC Metson
17,8 8,09 nd 10,7 19,9 12,6
+
(cmol /kg)
P O Olsen % nd 0,09 0,03 nd 0,26 nd
2 5
nd = not determined
CEC: Cation exchange capacity
A.2.3 Soil DNA extraction
The procedure used for soil DNA extraction follows this International Standard. Briefly, 1 ml of a solution
containing 100 mmol/l tris (pH 8,0), 100 mmol/l EDTA, 100 mmol/l NaCl, 1 % (mass fraction) polyvinylpyrrolidone,
and 2 % (mass fraction) sodium dodecyl sulfate was added to 250 mg of soil in a 2 ml mini-bead-beater tube
containing 0,5 g of 106-mm and two 2-mm-diameter glass beads. Samples were then homogenized for 30 s at
1 600g in a mini-bead-beater cell disruptor (6.1), after which the samples were centrifuged at 14 000g for 1 min
at 4 °C. The collected supernatants were incubated for 10 min on ice with 1/10 volume of 5 mol/l sodium acetate
8 © ISO 2012 – All rights reserved

ISO 11063:2012(E)
and centrifuged at 14 000g for 5 min. After precipitation with one volume of ice-cold isopropanol, the nucleic
acids were added with 70 % ethanol. Soil DNA extracts were then shipped to Laboratoire de Microbiologie du
Sol et de l’Environnement (Dijon, France), in dry ice.
It has to be noted that all the soil DNA extractions were done with two identical mini-bead beaters which were
shared between the different laboratories involved in this project.
A.2.4 Soil DNA purification
The procedure used for soil DNA purification consists of two steps:
a) an affinity column constituted of polyvinylpyrrolidone (PVPP) which specifically bounds humic acid substances;
1)
b) an exclusion column constituted of Sepharose 4B as described in Reference [20]. Briefly, PVPP-columns
were prepared by placing 92 mg to 95 mg (i.e. 1,2 cm) of PVPP powder into micro-spin chromatography
2)
columns after which 400 µl of H O were added and the tubes were centrifuged (2 min at 1 000g). The
procedure was repeated twice. For the Sepharose-columns, 1 ml of Sepharose 4B was placed in micro-
spin chromatography columns and centrifuged (2 min, 1 100g) after which the columns were washed by
the addition of 500 µl of TE buffer (tris-EDTA, pH 8) and centrifugation of the columns (2 min at 1 100g).
After preparation of the columns, soil DNA extracts were purified by centrifugation of the sample first
through the PVPP-column (1 000g for 4 min at 10 °C). The eluate was collected and was then passed
through a Sepharose-column by centrifugation at 1 500g for 4 min (10 °C).
A.2.5 Soil DNA quantification
The quantity of the extracted soil DNA was determined by resolved samples in 1 % agarose gels. After
3)
electrophoresis, gels were stained with ethidium bromide. Dilutions of calf tymus DNA were included in each
gel and a standard curve of DNA concentrations (60 ng, 30 ng, 15 ng, 7,5 ng and 3,25 ng) was used to estimate
4)
the quantity of DNA in the soil extracts. Obtained pictures of the gels were analysed using ImageQuaNT
software. The quantity of DNA was determined using the standard curve relating the intensity of DNA band to
the amount of DNA in ng. Yield of DNA extraction was calculated from the values as follows:
[yield of DNA, µg/g of soil] = DNA (ng/µl) × 0,4.
Data were analysed using the non-parametric Kruskal-Wallis test (p < 0,05).
A.2.6 Inhibition test
The presence of PCR inhibitors in the soil extracts was tested by the qPCR assay by mixing DNA extracts
5)
with control plasmid DNA (pGEM-T Easy eVector ). The test was performed on the purified soil DNA extracts
diluted at four different concentrations: 1 ng/µl, 0,5 ng/µl, 0,1 ng/µl and 0,01 ng/µl. The control plasmid was
[13]
amplified using universal primers Sp6 and T7 in accordance with the procedure described previously .
1) Sepharose 4B is the trade name of a product supplied by GE Healthcare Europe GmbH. This information is given
for the convenience of users of this International Standard 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.
2) Micro-spin chromatography columns are the trade name of a product supplied by Bio-Rad, USA. This information is
given for the convenience of users of this International Standard 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.
3) Calf tymus DNA is the trade name of a product supplied by Bio-Rad, USA. This information is given for the convenience
of users of this International Standard 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) ImageQuaNT is the trade name of a product supplied by Molecular Dynamics, Ca, USA. This information is given for the
convenience of users of this International Standard 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.
5) pGEM-T Easy Vector is the trade name of a product supplied by Promega, France. This information is given for the
convenience of users of this International Standard 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 11063:2012(E)
A.2.7 Quantitative PCR
The abundance of the total bacterial community was estimated by quantifying the amount of 16S rDNA
sequences with universal primers specific for eubacteria: 341f (5’-CCT ACG GGA GGC AGC AG-3’) and 534r
(5’-ATT ACC GCG GCT GCT GGC A-3’). Thermocycling conditions were as follows: 15 min at 95 °C; 30 cycles
consisting of 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C and 30 s at 80 °C.
Abundance of narG and pcaH bacterial communities was estimated by quantifying the amount of narG and
pcaH sequences, respectively. For narG amplification, degenerated primers narGf (5’-TCG CCS ATY CCG
[30]
GCS ATG TC-3’) and narGr (5’-GAG TTG TAC CAG TCR GCS GAY TCS G-3’) were used . Thermocycling
conditions were as follows: 15 min at 95 °C; 6 cycles consisting of 15 s at 95 °C, 30 s at 63 °C, with a touchdown
of 1 °C by cycle, and 30 s at 72 °C; 40 cycles consisting of 15 s at 95 °C, 30 s at 58 °C, 30 s at 72 °C, and 30 s
at 80 °C. For the amplification of pcaH sequences, degenerated primers pcaHf (5’-GAG RTS TGG CAR GCS
AAY-3’) and pcaHr (5’-CCG YSS AGC ACG ATG TC-3’) were used with the following thermocycling conditions:
15 min at 95 °C; 8 cycles consisting of 15 s at 95 °C, 30 s at 64 °C, with a touchdown of 0,5 °C by cycle, and
[9]
30 s at 72 °C; 30 cycles consisting of 15 s at 95 °C, 30 s at 60 °C, 30 s at 72 °C, and 30 s at 80 °C .
All qPCR assays were carried out in a total of 15 µl reaction volume containing SYBR green PCR Master Mix
6) 7)
(Absolute QPCR SYBR Green Rox ) 100 ng of T4 gp32 , 2 µl of template DNA (0,01 ng/µl) and 2 µmol/l
of each primer, except for the pcaH assay in which primers were used in different concentrations (pcaHf
2 µmol/l and pcaHr 1,2 µmol/l). qPCR assays were performed for each replicate (n = 5, n = 270). No-template
tot
controls were also included in all the assays. Standard curves for all the assays were obtained using tenfold
serial dilutions of a linearized plasmid pGEM-T (102 to 107 copies) containing 16S rRNA or narG sequence
originating from Pseudomonas aeruginosa PAO1 or, for the pcaH assay, pcaH sequence originating from the
environmental clone. Melting curves were generated after amplification by increasing the temperature from
80 °C to 95 °C.
The qPCR data were analysed using
a) multiple comparison parametric Tukey-Kramer test,
b) non-parametric Kruskal-Wallis test, and
c) Mantel test of correlation.
A.2.8 Automated RISA fingerprinting
Automated Ribosomal Intergenic Spacer Analysis (A-RISA) was used for studying the structure of bacterial
communities in the six soils under investigation in the international ring test. A-RISA was conducted on the
purified soil DNA diluted at 0,01 ng/µl. Targeted 16S–23S intergenic spacer of the bacterial rDNA was amplified
in a final volume of 25 µl with 0,5 µmol/l of A-RISA-1552f (5’-TCG GGC TGG ATG ACC TCC TT-3’) and A-RISA-
8)
132r (5’-CCG GGT TTC CCC ATT CGC -3’) universal primers, 2,5 U of Taq DNA polymerase and 2 µl of
9)
template DNA. The primer A-RISA-1552f was labelled in the 5’ position with IRD 800 dye fluorochrome . PCR
10)
amplification was carried out in a gradient thermocycler VentiTM with the following conditions: 5 min at 94 °C;
35 cycles of 1 min at 94 °C, 1 min at 55 °C and 2 min at 72 °C; plus an additional cycle for 15 min at 72 °C.
6) Absolute QPCR SYBR Green Rox is the trade name of a product supplied by ABgene, France. This information is given
for the convenience of users of this International Standard 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.
7) T4 gp32 is the trade name of a product supplied by QBiogene, France. This information is given for the convenience
of users of this International Standard 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.
8) Taq DNA polymerase is the trade name of a product supplied by Appligene Oncor, France. This information is given
for the convenience of users of this International Standard 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.
9) IRD 800 dye fluorochrome is the trade name of a product supplied by MWG SA Biotech, Ebersberg, Germany. This
information is given for the convenience of users of this International Standard 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.
10) VentiTM is the trade name of a product supplied by Applied Biosystems, USA. This information is given for the
convenience of users of this International Standard and does not constitute an endorsement by ISO of the product named.
10 © ISO 2012 – All rights reserved

ISO 11063:2012(E)
The quality of the A-RISA PCR products was checked by electrophoresis on 1 % agarose gel with Smart
11)
Ladder DNA marker, after which products were loaded on the 6,5 % polyacrylamide gels, (25 cm in length) and
12)
run on a LiCor 4 300 DNA Analysis System . Before loading on polyacrylamide gels samples were denatured
at 90 °C for 3 min. Separation of the A-RISA fragments were done during a 4 h run with electrophoresis
conditions as follows: 2 000 V, 25 mA and 45 W.
13)
The data were analysed using the 1D-Scan software . The software converted fluorescence data into
electrophoregrams where peaks represented PCR fragments. The height of the peaks was calculated in
conjunction with the median filter option and the Gaussian integration in 1D-Scan, and represented the relative
proportion of the fragments in the total products. Lengths (in base pairs) were calculated by using a size
standard with 15 bands ranging from 206 to 1 119 bp. Data from the 1D-Scan were converted into a matrix
summarizing the bands presence (i.e. peak) and intensity (i.e. height of peak) using PrepRISA program (http://
pbil.univ-lyon1.fr/ADE-4/microb/). Principal component analysis (PCA) on an A-RISA covariance matrix was
performed on the data using ADE-4 software (http://pbil.univ-lyon1.fr/ADE-4/home.php).
A.3 Results
A.3.1 Soil DNA extraction yields
The yield of DNA extracted by each of the nine participant laboratories from the six studied soils is presented
in Table A.2.
Table A.2 — Yield of soil DNA extracted from the six studied soils (µg of DNA/g of soil)
DNA concentration (µg/g soil)
Laboratory
A B C F M S
Sweden 3,53 ab 2,09 ab 0,44 a 4,21 b 1,75 a 1,48 b
France, Ineris 4,70 ab 1,23 a 1,09 abc 2,75 ab 3,47 ab 3,18 ab
Spain 1,01 a 2,45 ab 1,07 ab 2,88 ab 1,88 a 0,68 ab
Finland, Helsinki 3,98 ab 3,92 b 2,75 c nd 2,66ab 1,81 ab
France, Nancy 3,63 ab 2,46 ab 1,50 abc 3,05 ab 2,34 ab 1,39 ab
France, Dijon 6,75 b 2,47 ab 2,12 bc 3,17 ab 4,53 b 2,55 ab
Italy 3,21 ab 2,78 ab 0,95 ab 2,00 a 1,83 a 2,31 a
Germany, JKI 4,04 ab 3,53 b 2,00 bc 2,40 ab 2,51 ab 1,37 ab
Germany, Munich 0,98 a 2,94 ab 1,22 abc 3,59 ab 2,15 ab 0,93 ab
Letters (a, b, c) assigned to each value represent groups appointed by the Kruskal-Wallis statistical analysis (p < 0,05). Values in the
same group are not significantly different between each other.
nd = not determined
As can be seen from Table A.2, all nine laboratories were successful in extracting DNA from all studied soils,
likewise agricultural, forest and PAH-contaminated soil. Depending on the soil, results showed that DNA
quantities ranged in different samples from 0,44 µg (soil C extracted by the Swedish laboratory) to 6,75 µg
of DNA per gram of soil (so
...