1、May 2013 Translation by DIN-Sprachendienst.English price group 13No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 13
2、.080.30!%$D“2013398www.din.deDDIN EN ISO 11063Soil quality Method to directly extract DNA from soil samples (ISO 11063:2012);English version EN ISO 11063:2013,English translation of DIN EN ISO 11063:2013-05Bodenbeschaffenheit Verfahren zur direkten Extraktion von DNA aus Bodenproben (ISO 11063:2012)
3、;Englische Fassung EN ISO 11063:2013,Englische bersetzung von DIN EN ISO 11063:2013-05Qualit du sol Mthode pour extraire directement lADN dchantillons de sol (ISO 11063:2012);Version anglaise EN ISO 11063:2013,Traduction anglaise de DIN EN ISO 11063:2013-05www.beuth.deDocument comprises 29 pagesIn c
4、ase of doubt, the German-language original shall be considered authoritative.05.13 DIN EN ISO 11063:2013-05 2 A comma is used as the decimal marker. National foreword The text of ISO 11063:2012 has been prepared by Technical Committee ISO/TC 190 “Soil quality” and has been taken over as EN ISO 11063
5、:2013 by Technical Committee CEN/TC 345 “Characterization of soils” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Subcommittee NA 119-01-02-04 UA Biologische Verfahren of Working Comm
6、ittee NA 119-01-02 AA Abfall- und Bodenuntersuchung. Expert assistance and specialized laboratories will be required to perform the analyses described in this standard. The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 10381-6 DIN ISO 10381-
7、6 National Annex NA (informative) Bibliography DIN 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 EUROPEAN STANDARD NORME EUROPENNE E
8、UROPISCHE 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 directement lADN dchantillons de sol (ISO 11063:2012) Bodenbeschaffenheit - Verfahren zur direkten Extraktion
9、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.
10、 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 tr
11、anslation 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, Fin
12、land, 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 STANDARDIZAT
13、ION 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 reserved worldwide for CEN national Members. Ref. No. EN ISO 11063:2013: EDIN EN ISO 11063:2013-05 EN ISO 11063:2013
14、 (E) 2 Contents Page Foreword Introduction . 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle 5 5 Test materials .6 5.1 Soil 6 5.2 Chemicals .6 5.3 Buffers and reagents .7 6 Apparatus .8 7 Procedures .8 7.1 Preparation of soil samples 8 7.2 Mechanical and chemical lyses 8 7.
15、3 Protein precipitation 8 7.4 Nucleic acid precipitation and washing 8 7.5 Nucleic acid storage 8 8 Estimation of soil DNA quality and quantity .9 8.1 Soil DNA quality and purity 9 8.2 Soil DNA quantity .9 9 Validation of the extraction procedure 9 10 International ring test 9 11 Test report 9 Annex
16、 A (informative) International ring test for evaluating soil DNA extraction procedure .11 Bibliography 25 34Foreword 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
17、 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
18、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 C
19、EN-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, Hung
20、ary, 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 m
21、odification. DIN EN ISO 11063:2013-05 EN ISO 11063:2013 (E) 3 IntroductionDNA (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
22、 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 soi
23、l, 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 soil-extracted nucleic acids have provided a pertinent alternative to c
24、lassical 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 serve as genotypic (= molecular genetic) markers for determining micro
25、bial 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, erro
26、rs 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 this International Standard is to describe the procedure used to extra
27、ct 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) approach
28、es.DIN EN ISO 11063:2013-05 EN ISO 11063:2013 (E) 4 1 ScopeThis 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 ag
29、ricultural 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 microbi
30、al 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 micr
31、obiota 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
32、DNA extract.2 Normative referencesThe 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, Soi
33、l 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 laboratory3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1
34、soil DNADNA extracted from soil-living microorganisms and remaining DNA from dead microorganisms4 PrincipleDNA 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
35、 could be adapted (extraction from a 1 g soil sample) to assess the global structure of fungal communities32. 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 ar
36、e 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 w
37、ith 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.DIN EN ISO 11063:2013-05 EN ISO 11063:2013 (E) 5 5 Te
38、st materials5.1 SoilSoil 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
39、 only once. Some of these storage conditions are currently under testing.Figure 1 Schematic 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. 638
40、1-92 6).5.2.3 Sodium chloride, NaCl (CAS No. 7647-14-5).DIN EN ISO 11063:2013-05 EN ISO 11063:2013 (E) 6 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 Aceti
41、c acid or glacial acetic acid, CH3COOH (CAS No. 64-19-7).5.2.8. Isopropanol, 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 st
42、erilized (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 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
43、,0.5.3.3 NaCl, 1 mol/l, 58,44 g of NaCl in 1 000 ml of H2O.5.3.4 PVP 40, 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),
44、 100 ml of 1 mol/l NaCl, 50 ml of 20 % PVP 40, 100 ml of 20 % SDS in 450 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 %, 70
45、0 ml of pure ethanol in 300 ml of H2O.5.3.9 TE buffer, pH 8,0, 10 mmol/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 n
46、m.5.3.14 Pure DNA (100 ng/l)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 H2O.DIN EN ISO 11063:2013-05 EN ISO 11063:2013 (E) 7 5.3.16 TBE buffer 1, 100 ml of TBE buffer 10 in 900 ml of H2O.6 ApparatusUse standard laboratory equi
47、pment including pipettes, a centrifuge, fume hood cabinet, 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 quantifi
48、cation 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 sample in liquid nitrogen and keep it frozen
49、at 80 C until its use.7.2 Mechanical and chemical lysesAdd 0,5 g of 106 m 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.
