1、BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO 17601:2016)BS EN ISO 17601:2018EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM
2、 EN ISO 17601 February 2018 ICS 13.080.30 English Version Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO 17601:2016) Qualit du sol - Estimation de labondance de squences de gnes microbiens par amplification p
3、ar raction de polymrisation en chane (PCR) quantitative partir dADN directement extrait du sol (ISO 17601:2016) Bodenbeschaffenheit - Abschtzung der Hufigkeit ausgewhlter mikrobieller Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten (ISO 17601:2016) This European Standard was approved by
4、CEN on 14 February 2018. 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 stand
5、ards 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 no
6、tified 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,
7、Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-C
8、ENELEC Management Centre: Rue de la Science 23, B-1040 Brussels 2018 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 17601:2018 ENational forewordThis British Standard is the UK implementation of EN ISO 17601:2018. It is identi
9、cal to ISO 17601:2016. It supersedes BS ISO 17601:2016, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee EH/4, Soil quality.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purpo
10、rt to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2018 Published by BSI Standards Limited 2018ISBN 978 0 580 98302 3ICS 13.080.30Compliance with a British Standard cannot confer immunity from legal obligatio
11、ns.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 January 2016.Amendments/corrigenda issued since publicationDate Text affected31 March 2018 This corrigendum renumbers BS ISO 17601:2016 as BS EN ISO 17601:2018BRITISH STANDARDBS EN ISO 176
12、01:2018EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 17601 February 2018 ICS 13.080.30 English Version Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO 17601:2016) Qualit du sol - Estimation de labon
13、dance de squences de gnes microbiens par amplification par raction de polymrisation en chane (PCR) quantitative partir dADN directement extrait du sol (ISO 17601:2016) Bodenbeschaffenheit - Abschtzung der Hufigkeit ausgewhlter mikrobieller Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten
14、(ISO 17601:2016) This European Standard was approved by CEN on 14 February 2018. 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 b
15、ibliographical 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 res
16、ponsibility 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 R
17、epublic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROP
18、EN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels 2018 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 17601:2018 EBS EN ISO 17601:2018EN ISO 17601:2018 (E)
19、3 European foreword The text of ISO 17601:2016 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 17601:2018 by Technical Committee CEN/TC 444 “Test methods for environmental characterizatio
20、n 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 August 2018, and conflicting national standards shall be withdrawn at the latest by Au
21、gust 2018. 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. According to the CEN-CENELEC Internal Regulations, the national standards organizations of
22、 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, Neth
23、erlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 17601:2016 has been approved by CEN as EN ISO 17601:2018 without any modification. BS EN ISO 17601:2018ISO 17601:2016(E)Foreword ivInt
24、roduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Test materials 45.1 DNA . 45.2 Bacteria 45.3 Plasmid 45.4 Enzyme 45.5 Chemicals . 45.6 Product for bacterial culture medium 55.7 Buffer and reagents . 56 Apparatus . 67 Procedure. 67.1 qPCR standard preparation a
25、nd calibration of qPCR assay (task 1) 67.1.1 General 67.1.2 Amplicon design (task 1, step 1) 67.1.3 qPCR standard preparation (task 1, step 2) . 77.1.4 Isolate DNA, environmental DNA, artificial DNA 77.1.5 Calibration of the qPCR (task 1, step 3) . 97.2 Preparation of soil DNA template and inhibitio
26、n test (task 2) .107.2.1 General. 107.2.2 Soil DNA preparation (task 2, step 4) .107.2.3 Inhibition test (task 2, step 5) . 107.2.4 Dilution of DNA template 127.3 qPCR assay (task 3) 127.3.1 General. 127.3.2 qPCR 127.4 Validation and analysis of qPCR assay (task 4) 127.4.1 General. 127.4.2 Validatio
27、n of the qPCR assay 127.4.3 Calculation of the copy number of the gene of interest in the soil DNA extract.138 Examination of the critical steps of the qPCR assay 149 Expression of the results of the qPCR assay 1410 International ring test .1411 Test report 14Annex A (informative) Description of pri
28、ncipal steps of TaqManqPCR assay .15Annex B (informative) International ring-test for evaluating qPCR to quantify the abundance of selected microbial gene sequences from DNA directly extracted from soil 17Bibliography .30 ISO 2016 All rights reserved iiiContents PageBS EN ISO 17601:2018ISO 17601:201
29、6(E)ForewordISO (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
30、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 matt
31、ers 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 doc
32、ument 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 s
33、uch 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 do
34、es not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary inform
35、ationThe committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.iv ISO 2016 All rights reservedBS EN ISO 17601:2018ISO 17601:2016(E)IntroductionDNA (DNAs) is a major component of any living organisms coding for enzymes responsible for their biologi
36、cal activities. The study of DNA sequences from DNA sources extracted from different environmental 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, m
37、ost of the studies aiming to develop microbial quality indicators applicable to complex environment such as soil were biased by the poor culturability of many microorganisms under laboratory conditions and the lack of sensitivity of traditional microbiological methods. The recent development of a la
38、rge set of molecular biology methods based 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 of microbial communities.23456DNA-based approach
39、es are now well established in soil ecology and serve as genotypic markers for determining microbial diversity. The results of molecular analyzes of soil microbial communities and/or populations rely on two main parameters: a) the extraction of DNA representative of the indigenous bacterial communit
40、y composition and 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.7489Numerous studies have investigated new methods to improve extraction, purification, amplification, and quantification of DNA from soils.10Rec
41、ently, ISO 11063 reporting “a method to extract nucleic acids directly from soil samples“ derived from Reference 10 is opening a new window for developing standardized molecular approaches to estimate soil quality.11The aim of this International Standard is to describe the procedure used to set up a
42、nd perform quantitative PCR to quantify the abundance of soil microbial phyla, as well as functional groups from DNA directly extracted from soil samples. The quantification of soil microbial phyla, as well as functional groups by qPCR assays can contribute to the development of routine tools to mon
43、itor soil quality. The repeatability and the reproducibility of the procedure of the quantitative PCR were assessed in an international ring test study (see Annex B). The repeatability of this procedure was successfully evaluated for both 16S rRNA genes, as well as genes coding a functional marker o
44、f denitrifiers (the nitrite reductase gene nirK). The reproducibility of this procedure revealed a laboratory effect which can be overcome by interpreting the results of the quantification of the abundance of the microbial groups by comparison, either by using an external reference (DNA extracted fr
45、om a control strain) in the assay or by calculating a percentage of variations between treatments to normalize the data. ISO 2016 All rights reserved vBS EN ISO 17601:2018Soil quality Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from so
46、il1 ScopeThis International Standard specifies the crucial steps of a quantitative real-time polymerase chain reaction (qPCR) method to measure the abundance of selected microbial gene sequences from soil DNA extract which provides an estimation of selected microbial groups.It is noteworthy that the
47、 number of genes is not necessarily directly linked to the number of organisms that are measured. For example, the number of ribosomal operon is ranging from one copy to 20 copies in different bacterial phyla. Therefore, the number of 16S rRNA sequences quantified from soil DNA extracts does not giv
48、e an exact estimate of the number of soil bacteria. Furthermore, the number of sequences is not necessarily linked to living microorganisms and can comprise sequences amplified from dead microorganisms.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in
49、this document and are indispensable for its application. 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 laboratoryISO 11063, Soil quality Method to directly extract DNA from soil samples3 Terms an
