oSIST prEN ISO 17601:2024

Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO/DIS 17601:2024)

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/DIS 17601:2024)

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/DIS 17601:2024)

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/DIS 17601:2024)

General Information

• Status: Not Published
• Public Enquiry End Date: 01-Nov-2024
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: KAT - Soil quality
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 22-Aug-2024
• Due Date: 09-Jan-2025
• Completion Date: 04-Nov-2024
• Ref Project: prEN ISO 17601 - Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO/DIS 17601:2024)

Relations

• Revises: SIST EN ISO 17601:2018 - Soil quality - Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil (ISO 17601:2016)
• Effective Date: 01-Nov-2024

Standards Content (Sample)

SLOVENSKI STANDARD
01-oktober-2024
Kakovost tal - Ocena številčnosti izbranih sekvenc mikrobnih genov s
kvantitativnim PCR analizatorjem v talnih ekstraktih DNK (ISO/DIS 17601:2024)
Soil quality - Estimation of abundance of selected microbial gene sequences by
quantitative PCR from DNA directly extracted from soil (ISO/DIS 17601:2024)
Bodenbeschaffenheit- Ermittlung der Häufigkeit ausgewählter mikrobieller
Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten (ISO/DIS
17601:2024)
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/DIS 17601:2024)
Ta slovenski standard je istoveten z: prEN ISO 17601
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.

DRAFT
International
Standard
ISO/DIS 17601
ISO/TC 190/SC 4
Soil quality — Estimation of
Secretariat: AFNOR
abundance of selected microbial
Voting begins on:
gene sequences by quantitative PCR
2024-08-15
from DNA directly extracted from
Voting terminates on:
soil
2024-11-07
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
ICS: 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
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BEING ACCEPTABLE FOR INDUSTRIAL,
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USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
ISO/CEN PARALLEL PROCESSING
BE CONSIDERED IN THE LIGHT OF THEIR
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RECIPIENTS OF THIS DRAFT ARE INVITED
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PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 17601:2024(en)
DRAFT
ISO/DIS 17601:2024(en)
International
Standard
ISO/DIS 17601
ISO/TC 190/SC 4
Soil quality — Estimation of
Secretariat: AFNOR
abundance of selected microbial
Voting begins on:
gene sequences by quantitative PCR
from DNA directly extracted from soil
Voting terminates on:
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
ICS: 13.080.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2024
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
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BE CONSIDERED IN THE LIGHT OF THEIR
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NATIONAL REGULATIONS.
ISO copyright office
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Website: www.iso.org
Published in Switzerland Reference number
ISO/DIS 17601:2024(en)
ii
ISO/DIS 17601:2024(en)
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 .13
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
Annex C (informative) Examples of well established primer systems for a qPCR based
quantification of marker genes in soil samples .30
Bibliography .33

iii
ISO/DIS 17601:2024(en)
Foreword
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