1、BSI Standards PublicationBS ISO 17601:2016Soil quality Estimationof abundance of selectedmicrobial gene sequences byquantitative PCR from DNAdirectly extracted from soilBS ISO 17601:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 17601:2016. The UK partici
2、pation in its preparation was entrusted to TechnicalCommittee EH/4, Soil quality.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its co
3、rrect application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 76718 0 ICS 13.080.30 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and S
4、trategy Committee on 31 January 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 17601:2016 ISO 2016Soil quality Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soilQualit du sol Estimation de labo
5、ndance de squences de gnes microbiens par amplification par raction de polymrisation en chane (PCR) quantitative partir dADN directement extrait du solINTERNATIONAL STANDARDISO17601First edition2016-01-15Reference numberISO 17601:2016(E)BS ISO 17601:2016ISO 17601:2016(E)ii ISO 2016 All rights reserv
6、edCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intran
7、et, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.is
8、o.orgBS ISO 17601:2016ISO 17601:2016(E)Foreword ivIntroduction 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 Appar
9、atus . 67 Procedure. 67.1 qPCR standard preparation and 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)
10、. 97.2 Preparation of soil DNA template and inhibition 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 q
11、PCR assay (task 4) 127.4.1 General. 127.4.2 Validation 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 T
12、est report 14Annex A (informative) Description of principal 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 reser
13、ved iiiContents PageBS ISO 17601:2016ISO 17601:2016(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. E
14、ach 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 Internati
15、onal Electrotechnical Commission (IEC) on all matters 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 differen
16、t types of ISO documents should be noted. This document 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 no
17、t be held responsible for identifying any or all such 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 inf
18、ormation given for the convenience of users and does 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
19、the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological methods.iv ISO 2016 All rights reservedBS ISO 17601:2016ISO 17601:2016(E)IntroductionDNA (DNAs) is a major component of any living organisms c
20、oding for enzymes responsible for their biological 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 (
21、bacteria, archaea, and eucaryotes).Up to now, most 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 microbio
22、logical methods. The recent development of a large 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 o
23、f microbial communities.23456DNA-based approaches 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 repr
24、esentative of the indigenous bacterial community 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, amplificat
25、ion, and quantification of DNA from soils.10Recently, 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 Standar
26、d is to describe the procedure used to set up and 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 contri
27、bute to the development of routine tools to monitor 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
28、, as well as genes coding a functional marker of 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 b
29、y using an external reference (DNA extracted from 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 ISO 17601:2016BS ISO 17601:2016Soil quality Estimation of abundance of selected microbial gene sequ
30、ences by quantitative PCR from DNA directly extracted from soil1 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 esti
31、mation of selected microbial groups.It is noteworthy that the 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
32、rRNA sequences quantified from soil DNA extracts does not give 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 d
33、ocuments, in whole or in part, are normatively referenced in 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 quali
34、ty 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 and definitionsFor the purpose
35、s of this document, the following terms and definitions apply.3.1soil DNADNA extracted from soil of living and dead biotaEXAMPLE Microorganisms, plants, animals.3.2polymerase chain reactionPCRmethod allowing the amplification of a specific DNA sequence using a specific pair of oligonucleotide primer
36、s3.3quantitative polymerase chain reactionqPCRmethod allowing the quantification in a DNA template (3.4) of the number of a specific DNA sequence using a specific pair of oligonucleotide primers3.4templateDNA sample used to perform PCR (3.2) to amplify a specific DNA sequenceINTERNATIONAL STANDARD I
37、SO 17601:2016(E) ISO 2016 All rights reserved 1BS ISO 17601:2016ISO 17601:2016(E)3.5ampliconPCR product obtained by PCR (3.2) from a template (3.4)3.6cloning vectorcircular DNA molecule in which the amplicon (3.5) is inserted by ligation used to transform competent Escherichia coli for cloning the a
38、mplicon3.7qPCR standardcloned DNA target used as template (3.4) for qPCR reaction to establish the standard curve relating the abundance of target sequence as a function of cycle threshold values (Ct)3.8non-template controlNTCcontrol, usually molecular grade water, that is used as negative control i
39、n qPCR assay to check for the absence of contaminant in the qPCR mix3.9cycle thresholdCtnumber of qPCR cycles required for the fluorescent signal to cross the threshold (i.e. exceeds background level)Note 1 to entry: The Ct value is inversely proportional to the abundance of the target sequence.4 Pr
40、incipleThis International Standard describes qPCR assay using fluorescent DNA binding dye as reporter. This qPCR assay has been validated by an international ring test conducted with the SYBR Green, a commonly used fluorescent DNA binding dye which binds all doublestranded DNA and can be detected by
41、 measuring the increase in fluorescence throughout the cycle.The method aims to measure the abundance of selected microbial gene sequences from soil DNA extract. The method comprises four tasks and eight steps as summarized in Figure 1. According to Reference 1, the three critical steps to be valida
42、ted for each qPCR assay are as shown in Figure 1.2 ISO 2016 All rights reservedBS ISO 17601:2016ISO 17601:2016(E)ValidationValidationFigure 1 Main tasks and critical steps to estimate the abundance of selected microbial gene sequences by qPCR assayThis International Standard describes qPCR assay bas
43、ed on the use of fluorescent DNA binding dye which has been validated by an international ring test using SYBR Green1)qPCR. In Annex A, 1) SYBR Green is a registered trademark of Molecular Probes. This information is given for the convenience of users of this document and does not constitute an endo
44、rsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results. ISO 2016 All rights reserved 3BS ISO 17601:2016ISO 17601:2016(E)information about TaqMan2)qPCR assay not tested in the international ring test are given. The first task is made of t
45、hree steps describing the design of optimal amplicon for qPCR (step one), the preparation of qPCR standards (step two), and the procedure to calibrate the qPCR assay (step three). The second task includes two additional steps describing the procedures to prepare soil DNA samples (step four) and to t
46、est for the presence of qPCR inhibitors in soil DNA samples (step five). The third task is constituted of a single step describing the protocol to perform qPCR assay (step six). Finally, the fourth task is made of two steps, one describing the procedure to validate qPCR assays (step 7) to check the
47、quality of qPCR assay and another one describing the different options to calculate the number of sequences of the gene of interest copy from cycle threshold (Ct) obtained from the analysis of qPCR amplification plots (step 8).5 Test materials5.1 DNA5.1.1 DNA, extracted from pure bacterial and funga
48、l isolates using classical extraction procedures or by using commercial kit to extract genomic DNA.5.1.2 Soil DNA, extracted from aliquots of soil according to ISO 11063.5.2 Bacteria5.2.1 Escherichia coli strain, usually used for cloning of PCR product.5.3 Plasmid5.3.1 Cloning vector, usually used f
49、or cloning of PCR product in competent Escherichia coli.5.4 Enzyme5.4.1 Taq polymerase.5.4.2 T4 DNA ligase.5.4.3 T4 gene T32.5.4.4 Bovine serum albumin (CAS No. 9048-46-8).5.5 Chemicals5.5.1 Ampicilline sodium, C16H18N3NaO4S (CAS No. 69-52-3).5.5.2 Boric acid, BH3O3(CAS No. 10043-35-3).5.5.3 Deoxynucleotide solution, dNTPs.5.5.4 SYBR SafeDNA gel stain.2) TaqMan is a trademark of Roche Molecular Systems, Inc. This information is given for the convenience of users of this document and does not constitute an
