EN ISO 17601:2018
(Main)Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO 17601:2016)
Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO 17601:2016)
ISO 17601:2016 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 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 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.
Bodenbeschaffenheit - Ermittlung der Häufigkeit ausgewählter mikrobieller Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten (ISO 17601:2016)
Diese Internationale Norm legt die entscheidenden Schritte eines Verfahrens der quantitativen Real-time-Polymerase-Kettenreaktion (qPCR) zur Messung der Häufigkeit ausgewählter mikrobieller Gensequenzen aus Boden-DNA-Extrakten fest, welche eine Quantifizierung ausgewählter mikrobieller Gruppen ermöglicht.
Dabei gilt, dass die Anzahl von Genen nicht zwangsläufig direkt mit der Anzahl an gemessenen Organismen korreliert. Zum Beispiel wurden für ribosomale Operons in unterschiedlichen Bakterienstämmen von einer bis zu 20 Kopien pro Zelle beschrieben. Daher ergibt die Quantifizierung von 16S rRNA-Sequenzen aus extrahierter DNA aus Bodenproben mittels qPCR nur einen Anhaltspunkt für die Häufigkeit von Bodenbakterien. Außerdem ist die DNA auch nach dem Zelltod in Böden persistent und daher kein Maß für die lebende Biomasse.
Qualité du sol - Estimation de l'abondance de séquences de gènes microbiens par amplification par réaction de polymérisation en chaîne (PCR) quantitative à partir d'ADN directement extrait du sol (ISO 17601:2016)
ISO 17601:2016 spécifie les étapes principales d'une méthode d'amplification par réaction de polymérisation en chaîne (PCR) quantitative (qPCR) permettant de mesurer l'abondance de séquences spécifiques de gènes microbiens à partir d'un extrait d'ADN du sol qui fournit une estimation de l'abondance de groupes microbiens spécifiques.
Il convient de noter que le nombre de gènes n'est pas nécessairement lié directement au nombre de micro-organismes mesurés. Par exemple, le nombre d'opérons ribosomiques est compris entre une et 20 copies dans différents phyla bactériens. Par conséquent, le nombre de séquences d'ARNr 16S quantifiées dans des extraits d'ADN du sol ne donne pas une estimation exacte du nombre de bactéries contenues dans le sol. Par ailleurs, le nombre de séquences n'est pas nécessairement lié à des micro-organismes vivants et peut comprendre des séquences amplifiées à partir de l'ADN extrait de micro-organismes morts.
Kakovost tal - Ocena številčnosti izbranih sekvenc mikrobnih genov s kvantitativnim PCR analizatorjem v talnih ekstraktih DNK (ISO 17601:2016)
Standard ISO 17601:2016 določa ključne korake pri metodi kvantitativne verižne reakcije s polimerazo v realnem času (qPCR) za merjenje številčnosti izbranih sekvenc mikrobnih genov v talnih ekstraktih DNK, ki zagotavlja oceno izbranih mikrobnih skupin.
Omeniti je treba, da število genov ni nujno neposredno povezano s številom organizmov, ki so izmerjeni. Število ribosomskih operonov lahko na primer znaša od ene do 20 kopij v različnih deblih bakterij. Zaradi tega število sekvenc 16S rRNA, kvantificiranih iz talnih ekstraktov DNK, ne zagotavlja točne ocene števila bakterij v tleh. Poleg tega število sekvenc ni nujni povezano z živimi mikroorganizmi in lahko zajema sekvence, povečane z mrtvimi mikroorganizmi.
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
01-julij-2018
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NYDQWLWDWLYQLP3&5DQDOL]DWRUMHPYWDOQLKHNVWUDNWLK'1. ,62
Soil quality - Estimation of abundance of selected microbial gene sequences by
quantitative PCR from DNA directly extracted from soil (ISO 17601:2016)
Bodenbeschaffenheit - Abschätzung der Häufigkeit ausgewählter mikrobieller
Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten (ISO 17601:2016)
Qualité du sol - Estimation de l'abondance de séquences de gènes microbiens par
amplification par réaction de polymérisation en chaîne (PCR) quantitative à partir d'ADN
directement extrait du sol (ISO 17601:2016)
Ta slovenski standard je istoveten z: EN ISO 17601:2018
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.
EN ISO 17601
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2018
EUROPÄISCHE NORM
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)
Qualité du sol - Estimation de l'abondance de Bodenbeschaffenheit - Abschätzung der Häufigkeit
séquences de gènes microbiens par amplification par ausgewählter mikrobieller Gensequenzen durch
réaction de polymérisation en chaîne (PCR) quantitative PCR aus DNA-Boden-Extrakten (ISO
quantitative à partir d'ADN directement extrait du sol 17601:2016)
(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 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR 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 Ref. No. EN ISO 17601:2018 E
worldwide for CEN national Members.
Contents Page
European foreword . 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 characterization 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 August 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 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, 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.
INTERNATIONAL ISO
STANDARD 17601
First edition
2016-01-15
Soil quality — Estimation of
abundance of selected microbial gene
sequences by quantitative PCR from
DNA directly extracted from soil
Qualité du sol — Estimation de l’abondance de séquences de gènes
microbiens par amplification par réaction de polymérisation en
chaîne (PCR) quantitative à partir d’ADN directement extrait du sol
Reference number
ISO 17601:2016(E)
©
ISO 2016
ISO 17601:2016(E)
© ISO 2016, Published in Switzerland
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ii © ISO 2016 – All rights reserved
ISO 17601:2016(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Test materials . 4
5.1 DNA . 4
5.2 Bacteria . 4
5.3 Plasmid . 4
5.4 Enzyme . 4
5.5 Chemicals . 4
5.6 Product for bacterial culture medium . 5
5.7 Buffer and reagents . 5
6 Apparatus . 6
7 Procedure. 6
7.1 qPCR standard preparation and calibration of qPCR assay (task 1) . 6
7.1.1 General. 6
7.1.2 Amplicon design (task 1, step 1) . 6
7.1.3 qPCR standard preparation (task 1, step 2) . 7
7.1.4 Isolate DNA, environmental DNA, artificial DNA . 7
7.1.5 Calibration of the qPCR (task 1, step 3) . 9
7.2 Preparation of soil DNA template and inhibition test (task 2) .10
7.2.1 General.10
7.2.2 Soil DNA preparation (task 2, step 4) .10
7.2.3 Inhibition test (task 2, step 5) .10
7.2.4 Dilution of DNA template .12
7.3 qPCR assay (task 3) .12
7.3.1 General.12
7.3.2 qPCR .12
7.4 Validation and analysis of qPCR assay (task 4) .12
7.4.1 General.12
7.4.2 Validation of the qPCR assay .12
7.4.3 Calculation of the copy number of the gene of interest in the soil DNA extract.13
8 Examination of the critical steps of the qPCR assay .14
9 Expression of the results of the qPCR assay .14
10 International ring test .14
11 Test report .14 ®
Annex A (informative) Description of principal steps of TaqMan qPCR assay .15
Annex B (informative) International ring-test for evaluating qPCR to quantify the
abundance of selected microbial gene sequences from DNA directly extracted from soil .17
Bibliography .30
ISO 17601:2016(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,
...
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