1、BSI Standards PublicationBS EN ISO 11063:2013Soil quality Method todirectly extract DNA from soilsamples (ISO 11063:2012)BS EN ISO 11063:2013 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO11063:2013. It is identical to ISO 11063:2012.The UK participation in
2、 its preparation was entrusted to TechnicalCommittee EH/4, Soil quality.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplic
3、ation. The British Standards Institution 2013. Published by BSI StandardsLimited 2013ISBN 978 0 580 79339 4ICS 13.080.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committe
4、e on 30 November 2013.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 11063 February 2013 ICS 13.080.30 English Version Soil quality - Method to directly extract DNA from soil samples (ISO 11063:2012) Qualit du sol - Mthode pour extraire
5、directement lADN dchantillons de sol (ISO 11063:2012) Bodenbeschaffenheit - Verfahren zur direkten Extraktion 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 stipul
6、ate 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 St
7、andard 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 n
8、ational 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,
9、Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserv
10、ed worldwide for CEN national Members. Ref. No. EN ISO 11063:2013: EBS EN ISO 11063:2013EN ISO 11063:2013 (E) 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 a
11、s 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 conflict
12、ing 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 t
13、he 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,
14、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 a
15、ny modification. BS EN ISO 11063:2013ISO 11063:2012(E) ISO 2012 All rights reserved iiiContents PageForeword ivIntroduction v1 Scope 12 Normative references . 13 Terms and definitions . 14 Principle . 15 Test materials . 25.1 Soil . 25.2 Chemicals 25.3 Buffers and reagents 36 Apparatus 47 Procedures
16、 47.1 Preparation of soil samples 47.2 Mechanical and chemical lyses . 47.3 Protein precipitation . 47.4 Nucleic acid precipitation and washing 47.5 Nucleic acid storage . 48 Estimation of soil DNA quality and quantity 58.1 Soil DNA quality and purity. 58.2 Soil DNA quantity 59 Validation of the ext
17、raction procedure 510 International ring test . 511 Test report . 5Annex A (informative) International ring test for evaluating soil DNA extraction procedure 7Bibliography .21BS EN ISO 11063:2013ISO 11063:2012(E)ForewordISO (the International Organization for Standardization) is a worldwide federati
18、on 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
19、. 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 wi
20、th 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 approv
21、al 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
22、ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.iv ISO 2012 All rights reservedBS EN ISO 11063:2013ISO 11063:2012(E)IntroductionDNA (deoxyribonucleic acid) is an essential component of any living organism coding for enzymes responsible for any biological activities. The study of DNA
23、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 s
24、oil quality indicators applicable to complex environments, such as soil, were biased by the unculturability of many microorganisms and the lack of sensitivity of traditional microbiological methods 16. The recent development of numerous molecular biology methods based primarily on amplification of s
25、oil-extracted nucleic acids have provided a pertinent alternative to classical culture-based microbiological methods, providing unique insight into the composition, richness, and structure of microbial communities 15, 18, 26, 27, 36. DNA-based approaches are now well-established in soil ecology and
26、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, s
27、uch as the choice of primers, the concentration of amplified DNA, errors in the PCR, or even the method chosen for analysis 23, 26, 38, 40. Recently, numerous studies have investigated new methods to improve extraction, purification, amplification, and quantification of DNA from soils 20.The aim of
28、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 ev
29、aluated on both quantitative (q-PCR) and qualitative (A-RISA) approaches. ISO 2012 All rights reserved vBS EN ISO 11063:2013BS EN ISO 11063:2013Soil quality Method to directly extract DNA from soil samples1 ScopeThis International Standard specifies a method for direct extraction of DNA from soil sa
30、mples 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
31、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 reacti
32、on) 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
33、 (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 referencesThe following referenced documents are indispensable for the application of this document. For dated references,
34、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
35、processes, biomass and diversity in the laboratory3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1soil DNADNA extracted from soil-living microorganisms and remaining DNA from dead microorganisms4 PrincipleDNA is directly extracted from 0,25 g so
36、il samples using the following extraction procedure. This method reliably allowed analysing the global structure of bacterial and archeal communities and could be adapted (extraction from a 1 g soil sample) to assess the global structure of fungal communities32. Soil samples added with an extraction
37、 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 centrifugati
38、on, 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 i
39、s estimated using a spectro-fluorimeter. A schematic overview of the procedure is given in Figure 1.INTERNATIONAL STANDARD ISO 11063:2012(E) ISO 2012 All rights reserved 1BS EN ISO 11063:2013ISO 11063:2012(E)5 Test materials5.1 SoilSoil samples should be collected and sieved (2 mm mesh). If samples
40、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 Schema
41、tic overview of soil DNA extraction procedure5.2 Chemicals5.2.1 Trishydroxymethylaminomethane, C4H11NO3(CAS No. 77-86-1).5.2.2 Ethylenediaminetetraacetic acid disodium salt (EDTA), C10H14N2O8Na22 H2O (CAS No. 6381-92 6).5.2.3 Sodium chloride, NaCl (CAS No. 7647-14-5).2 ISO 2012 All rights reservedBS
42、 EN ISO 11063:2013ISO 11063:2012(E)5.2.4 Sodium dodecyl sulfate (SDS), CH3(CH2)11OSO3Na (CAS No. 151-21-3).5.2.5 Polyvinylpyrrolidone (PVP), C6H9NOn(CAS No. 9003-39-8).5.2.6 Sodium acetate, CH3COONa (CAS No. 6131-90-4).5.2.7 Acetic acid or glacial acetic acid, CH3COOH (CAS No. 64-19-7).5.2.8. Isopro
43、panol, CH3CHOHCH3(CAS No. 67-63-0).5.2.9 Ethanol, CH3CH2OH (CAS No. 64-17-5).5.2.10 Molecular-biology-grade water, H2O.5.3 Buffers and reagentsBuffers and reagents (except intercalent molecules) used for soil DNA extraction are sterilized (120 C for 20 min) and stored at room temperature. Ethanol an
44、d isopropanol are stored at 20 C.5.3.1 Tris-HCl, 1 mol/l, 121,14 g of tris in 1 000 ml of H2O, 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 H2O, 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 H2O.5.3.4 PVP 4
45、0, 20 %, 200 g of PVP in 1 000 ml of H2O.5.3.5 SDS, 20 %, 200 g of SDS in 1 000 ml of H2O.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 4
46、50 ml of H2O.5.3.7 Sodium acetate, 5 mol/l (pH 5,5), 410,15 g of CH3COONa in 800 ml of H2O. Add 120 ml of acetic acid 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 H2O.5.3.9 TE buffer, pH 8,0, 10 mmol
47、/l tris-HCl, 1 mmol/l EDTA.5.3.10 Glass beads (106 m).5.3.11 Glass beads (2 mm).5.3.12 Ethidium bromide, 5 mg of ethidium bromide in 1 000 ml of H2O.5.3.13 Fluorescent nucleic acid stain, excitation at 480 nm and emission at 520 nm.5.3.14 Pure DNA (100 ng/l)5.3.15 TBE buffer 10, pH 8,0, 108 g of tri
48、s base, 55 g of boric acid, 40 ml of 0,5 mol/l EDTA (pH 8,0) in 1 000 ml of H2O. ISO 2012 All rights reserved 3BS EN ISO 11063:2013ISO 11063:2012(E)5.3.16 TBE buffer 1, 100 ml of TBE buffer 10 in 900 ml of H2O.6 ApparatusUse standard laboratory equipment including pipettes, a centrifuge, fume hood c
49、abinet, horizontal electrophoresis system and the following.6.1 Mini-bead beating apparatus, with a beating frequency varying from, for example, 100 min-1to 2 600 min-1and 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 Procedures7.1 Preparation of soil samplesWeigh 0,25 g of soil (equivalent dry mass) in 2 ml micro-tubes just before extracting, or immediately freeze the soil samp
