ISO 17601:2016 PDF

Soil quality — Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil

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Title (English):

ISO 17601:2016

Soil quality — Estimation of abundance of selected microbial gene sequences by quantitative PCR from DNA directly extracted from soil

Document status:: Withdrawn
Format:: Electronic (PDF)
Number of pages:: 31
Publication date:: January 12, 2016
Edition:: ISO IS 17601 edition 1 version 1
ICS:: 13.080.30

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Description (English):

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.

Technical details

Technical committee: ISO/TC 190/SC 4 - Biological characterization
SKU: ISO 17601:2016

Abstract

Overview

ISO 17601:2016 - Soil quality specifies a standardized workflow to estimate the abundance of selected microbial gene sequences using quantitative real-time PCR (qPCR) on DNA directly extracted from soil. The standard describes the critical steps from amplicon design and qPCR standard preparation to soil DNA preparation, inhibition testing, qPCR assay execution (SYBR Green® and TaqMan® options) and validation. It emphasises that quantified gene copy numbers (e.g., 16S rRNA sequences) are proxies for microbial groups and are not necessarily equivalent to live organism counts due to variable gene copy numbers and DNA from dead cells.

Key topics and technical requirements

Method principle: Four main tasks - (1) qPCR standard preparation and calibration, (2) preparation of soil DNA template and inhibition testing, (3) qPCR assay performance, (4) validation and result calculation.
Amplicon design: Selection of target gene sequences and primer/probe design tailored for selected microbial groups or functional genes.
Standards and calibration: Preparation of cloned amplicons or plasmid standards, calibration curves, and limit-of-detection considerations.
Soil DNA handling: Use of DNA extracted according to ISO 11063 and sample handling guidance (e.g., ISO 10381-6); checks for qPCR inhibitors common in soil extracts.
qPCR chemistries: Procedures for SYBR Green and TaqMan assays are provided in informative annexes.
Instrumentation & reagents: Requirements include a real-time qPCR instrument (single-copy detection sensitivity), spectrophotometer/spectrofluorimeter for DNA quantification, polymerases, plasmid/cloning vectors, and common molecular-biology reagents.
Validation & reporting: Criteria for assay validation, calculation of gene copy numbers from Ct values, and recommended test report elements. An international ring test (Annex A) is referenced for reproducibility assessment.
Limitations: Explicit note that gene copy numbers do not directly equate to organism numbers or viability.

Applications

ISO 17601:2016 is applicable to:

Environmental and soil-quality laboratories performing molecular microbial monitoring.
Agricultural research and agronomy for soil microbiome assessment.
Environmental impact assessments and remediation monitoring.
Academic and industrial research studying microbial community structure or specific functional genes.
Regulatory bodies and standard-setting organizations needing reproducible qPCR-based microbial indicators.

Related standards

ISO 11063 - Method to directly extract DNA from soil samples (referenced for soil DNA extraction).
ISO 10381-6 - Guidance on collection, handling and storage of soil for microbiological assessment.

Keywords: ISO 17601:2016, soil quality, qPCR, quantitative PCR, soil DNA, microbial gene sequences, 16S rRNA, SYBR Green, TaqMan, ISO 11063, soil microbiome, environmental monitoring.

Related standards

Mentioned in the description and other ISO standards

Abstract

Overview

Key topics and technical requirements

Method principle: Four main tasks - (1) qPCR standard preparation and calibration, (2) preparation of soil DNA template and inhibition testing, (3) qPCR assay performance, (4) validation and result calculation.
Amplicon design: Selection of target gene sequences and primer/probe design tailored for selected microbial groups or functional genes.
Standards and calibration: Preparation of cloned amplicons or plasmid standards, calibration curves, and limit-of-detection considerations.
Soil DNA handling: Use of DNA extracted according to ISO 11063 and sample handling guidance (e.g., ISO 10381-6); checks for qPCR inhibitors common in soil extracts.
qPCR chemistries: Procedures for SYBR Green and TaqMan assays are provided in informative annexes.
Instrumentation & reagents: Requirements include a real-time qPCR instrument (single-copy detection sensitivity), spectrophotometer/spectrofluorimeter for DNA quantification, polymerases, plasmid/cloning vectors, and common molecular-biology reagents.
Validation & reporting: Criteria for assay validation, calculation of gene copy numbers from Ct values, and recommended test report elements. An international ring test (Annex A) is referenced for reproducibility assessment.
Limitations: Explicit note that gene copy numbers do not directly equate to organism numbers or viability.

Applications

ISO 17601:2016 is applicable to:

Environmental and soil-quality laboratories performing molecular microbial monitoring.
Agricultural research and agronomy for soil microbiome assessment.
Environmental impact assessments and remediation monitoring.
Academic and industrial research studying microbial community structure or specific functional genes.
Regulatory bodies and standard-setting organizations needing reproducible qPCR-based microbial indicators.

Related standards

ISO 11063 - Method to directly extract DNA from soil samples (referenced for soil DNA extraction).
ISO 10381-6 - Guidance on collection, handling and storage of soil for microbiological assessment.

Keywords: ISO 17601:2016, soil quality, qPCR, quantitative PCR, soil DNA, microbial gene sequences, 16S rRNA, SYBR Green, TaqMan, ISO 11063, soil microbiome, environmental monitoring.

ISO 17601:2016 PDF

Technical details

Abstract

Overview

Key topics and technical requirements

Applications

Related standards

Related standards

Abstract

Overview

Key topics and technical requirements

Applications

Related standards

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