kSIST FprEN ISO 17601:2025

SLOVENSKI STANDARD
oSIST prEN ISO 17601:2024
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
oSIST prEN ISO 17601:2024 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN ISO 17601:2024
oSIST prEN ISO 17601:2024
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
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This document is circulated as received from the committee secretariat.
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PROVIDE SUPPORTING DOCUMENTATION.
Reference number
ISO/DIS 17601:2024(en)
oSIST prEN ISO 17601:2024
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
ISO/CEN PARALLEL PROCESSING
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BE CONSIDERED IN THE LIGHT OF THEIR
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TO SUBMIT, WITH THEIR COMMENTS,
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NOTIFICATION OF ANY RELEVANT PATENT
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Website: www.iso.org
Published in Switzerland Reference number
ISO/DIS 17601:2024(en)
ii
oSIST prEN ISO 17601:2024
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
oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
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, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International 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 different 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 not 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 information 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 ISO's adherence to the WTO principles in the Technical Barriers to Trade (TBT)
see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
methods.
This second edition cancels and replaces the first edition (ISO 17601:2016).
The main changes are as follow:
— Annex C has been expanded by adding examples of well established qPCR systems to quantify certain
microbial groups or their function.

iv
oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
Introduction
DNA (DNAs) is a major component of any living organisms coding 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 (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 microbiological 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
[2][3][4][5][6]
the composition, richness, and structure of microbial communities . DNA-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 representative 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
[7][4][8][9]
method chosen for analysis .
Numerous studies have investigated new methods to improve extraction, purification, amplification, and
[10]
quantification of DNA from soils . Recently, 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
[11]
molecular approaches to estimate soil quality .
The aim of this International Standard 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 contribute 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, 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 by 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.

v
oSIST prEN ISO 17601:2024
oSIST prEN ISO 17601:2024
DRAFT International Standard ISO/DIS 17601:2024(en)
Soil quality — Estimation of abundance of selected microbial
gene sequences by quantitative PCR from DNA directly
extracted from soil
1 Scope
This 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 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.
A list of currently well established qPCR Assays to assess selected functional traits of the soil microbiome is
listed in Annex C.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 108400, 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 laboratory
ISO 11063, Soil quality — Direct extraction of soil DNA
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
soil DNA
DNA extracted from soil of living and dead biota
EXAMPLE Microorganisms, plants, animals.
3.2
polymerase chain reaction
PCR
method allowing the amplification of a specific DNA sequence using a specific pair of oligonucleotide primers

oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
3.3
quantitative polymerase chain reaction
qPCR
method allowing the quantification in a DNA template (3.4) of the number of a specific DNA sequence using a
specific pair of oligonucleotide primers
3.4
template
DNA sample used to perform PCR (3.2) to amplify a specific DNA sequence
3.5
amplicon
PCR product obtained by PCR (3.2) from a template (3.4)
3.6
cloning vector
circular DNA molecule in which the amplicon (3.5) is inserted by ligation used to transform competent
Escherichia coli for cloning the amplicon
3.7
qPCR standard
cloned 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)
3.8
non-template control
NTC
control, usually molecular grade water, that is used as negative control in qPCR assay to check for the
absence of contaminant in the qPCR mix
3.9
cycle threshold
Ct
number 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 Principle
This 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 double–stranded DNA and can be detected by 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 validated for each qPCR assay are as shown in Figure 1.

oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
Figure 1 — Main tasks and critical steps to estimate the abundance of selected microbial gene
sequences by qPCR assay
oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
This International Standard describes qPCR assay based on the use of fluorescent DNA binding dye which
®1)
has been validated by an international ring test using SYBR Green qPCR. In Annex A, information about
®2)
TaqMan qPCR assay not tested in the international ring test are given. The first task is made of three
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 test 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 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 materials
5.1 DNA
5.1.1 DNA, extracted from pure bacterial and fungal 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 Bacteria
5.2.1 Escherichia coli strain, usually used for cloning of PCR product.
5.3 Plasmid
5.3.1 Cloning vector, usually used for cloning of PCR product in competent Escherichia coli.
5.4 Enzyme
5.4.1 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 Chemicals
5.5.1 Ampicilline sodium, C H N NaO S (CAS No. 69-52-3).
16 18 3 4
5.5.2 Boric acid, BH O (CAS No. 10043-35-3).
3 3
5.5.3 Deoxynucleotide solution, dNTPs.
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 endorsement by ISO of the product named. Equivalent products may be used if
they can be shown to lead to the same results.
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 endorsement by ISO of the product named. Equivalent products may be used if they
can be shown to lead to the same results.

oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en) ®
5.5.4 SYBR Safe DNA gel stain.
5.5.5 Ethylenediaminetetraacetic acid disodium salt (EDTA), C H N O Na ·2 H O (CAS No. 6381-92 6).
10 14 2 8 2 2
5.5.6 Glucose, C H O (CAS No. 50-99-7).
6 12 6
5.5.7 Chlorhydric acid, HCl (CAS No. 7647-01-0).
5.5.8 IPTG, Isopropyl-Beta-D-Thiogalactopyranoside, (CAS No. 367-93-1).
5.5.9 Magnesium chloride, MgCl (CAS No. 7786-30-3).
5.5.10 Magnesium sulfate, MgSO (CAS No. 7487-88-9).
5.5.11 Molecular-biology-grade water, H O.
5.5.12 Potassium chloride, KCl (CAS No. 7447-40-7).
5.5.13 Sodium chloride, NaCl (CAS No. 7647-14-5).
5.5.14 Tris[hydroxymethyl]aminomethane, C H NO (CAS No. 77-86-1).
4 11 3
5.5.15 X-Gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, (CAS No. 7240-90-6).
5.6 Product for bacterial culture medium
®3)
5.6.1 Bacto tryptone , enzymatic digest of casein.
5.6.2 Yeast extract powder (CAS No. 8013-01-2).
5.7 Buffer and reagents
5.7.1 Ampicilline solution, 2 g of ampicilline sodium in 4 ml of 0,22 µm filter sterilized H O. Adjust to
20 ml with sterilized H O, prepare 1 ml aliquots, and store at −20 °C.
−1 −1
5.7.2 EDTA, 0,5 mol·l , 186,10 g of EDTA in 1 000 ml of H O adjusting with NaOH (10 mol·l ) to pH 8,0.
5.7.3 SYBR Safe™ DNA gel stain, dilute 10,000X SYBR Safe™ gel stain in TBE buffer × 1.
5.7.4 IPTG stock solution, 1 g of IPTG in 8 ml of H O. After careful mixing, the solution is adjusted to
10 ml and sterilized under security microbiology post. Prepare 1 ml aliquot of IPTG and store at −20 °C. ®
5.7.5 Solid LB medium, 10 g of Bacto tryptone , 5 g of yeast extract, 5 g of sodium chloride, and 15 g
of agar in 1 000 ml of H O. After autoclaving for 20 min at 120 °C, 1 ml of ampicilline stock solution at
−1
100 mg·ml is added to LB medium and plated in Petri dishes (20 ml) under a security microbiology post.
100 µl of IPTG solution are plated on solid LB-ampicilline medium. When IPTG solution is entered in LB-
ampicilline medium, 20 µl of X-Gal solution is plated on solid LB-ampicilline medium. Solid LB medium is
stored at 4 °C until its use.
3) Bacto tryptone is the trademark of a product supplied by Difco Laboratories. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent
products may be used if they can be shown to lead to the same results.

oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en) ®
5.7.6 SOC medium, 20 g of Bacto tryptone , 5 g of yeast extract, 0,58 g of NaCl, 0,95 g of MgCl , 2,46 g of
MgSO , and 3,60 g of glucose in 1 l H O. Sterilize by 20 min autoclaving at 120 °C. Prepare 950 ml aliquots
4 2
and store at −20 °C.
−1 −1
5.7.7 Tris-HCl, 1 mol·l , 121,14 g of Tris in 1 000 ml of H O adjusting with 4 mol·l HCl to pH 8,0.
−1
TBE buffer × 10, pH 8,0, 108 g of Tris base, 55 g of boric acid, and 40 ml of 0,5 mol·l EDTA (pH 8,0) in
1 000 ml of H O.
5.7.8 TBE buffer × 1, 100 ml of TBE buffer × 10 in 900 ml of H O.
−1 −1
5.7.9 TE buffer × 10, pH 8,0, 100 ml of 1 mol·l Tris-HCl pH 8,0, 20 ml of 50 mmol·l EDTA pH 8,0 in 880 ml
of molecular grade water.
5.7.10 TE buffer × 1, 100 ml of TE buffer × 10 in 900 ml of H O.
5.7.11 X-gal solution, 250 mg of X-Gal in 5 ml of dimethylformamide 5 ml. After careful mixing, prepare
0,5 ml aliquot and store at −20 °C.
6 Apparatus
Use standard laboratory equipment including pipettes, a centrifuge, fume hood cabinet, horizontal
electrophoresis system and the following.
6.1 Quantitative PCR, allowing the real-time quantification of amplicons from various DNA templates
with a theoretical detection limit of one copy of a sequence target per sample analyzed.
6.2 Spectro-photometer, allowing the quantification of double-strand DNA at 260 nm.
6.3 Spectro-fluorimeter, allowing the quantification of double-strand DNA.
NOTE Only one of these two apparatus is required to estimate DNA concentration.
7 Procedure
7.1 qPCR standard preparation and calibration of qPCR assay (task 1)
7.1.1 General
qPCR assay is based on the quantification of the amplicons at the end of each PCR cycle by using a DNA dye
which fluoresces when intercalated in the double strand amplicons. The purpose of this task is to describe
the definition of appropriate amplicon to settle down a qPCR assay (step one), the preparation of qPCR
standard (step two), and the calibration of the qPCR assay (step three).
7.1.2 Amplicon design (task 1, step 1)
7.1.2.1 General
The first step aims at designing oligonucleotide primer pair; it can be designed in silico using different
programs using the sequence of the microbial gene of interest to be quantified by qPCR from soil DNA extracts.
The specificity of the primers shall be checked in silico by comparing their sequences to known sequences
available in the Genbank database (http:// www .ncbi .nlm .nih .gov/ genbank/ ). Only primers specific for the
gene target shall be considered. The main parameters to be considered to design oligonucleotide primer pair
for establishing qPCR assay are listed thereafter.

oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
7.1.2.2 qPCR
— Optimal amplicon length ranges between 100 bp to 250 bp.
— Optimal primer length ranges between 18 bp and 25 bp with a GC content of 50 % and melting temperature
between 58 °C and 65 °C.
— The five nucleotides at the 3” end of each primer should have no more than two G and/or C bases.
— Avoid succession of identical nucleotide, especially true for guanine.
— 3” self-complementarity of the primer taken as a measure of its tendency to form a primer-dimer with
itself should be checked and avoided.
— Avoid design of primers with more than four mismatches because too high degeneracy of the primer
contributes to fluctuation of qPCR results.
7.1.3 qPCR standard preparation (task 1, step 2)
Step 2 of task 1 describes the procedure used to generate qPCR standards targeting a sequence of the
microbial gene of interest from different DNA templates (pure bacterial or fungal isolate, environmental
DNA, or artificial DNA). It also reports the procedure used to insert the qPCR standard in a cloning vector,
transform Escherichia coli, and purify recombinant plasmids harboring qPCR standard for further use for
qPCR assays.
7.1.4 Isolate DNA, environmental DNA, artificial DNA
7.1.4.1 General
The first step of qPCR standard preparation relies on the extraction of DNA templates known to harbour the
microbial gene of interest. This can be done starting from different materials such as the following:
a) pure cultures of microorganisms;
DNA is extracted from cells harvested from a fresh culture of microorganisms by using conventional
genomic DNA extraction protocols.
b) artificial DNA.
If no biological samples are available or known to harbour the gene of interest, artificial DNA made of the
sequence of the gene of interest can be synthesized.
In all cases, the quality of DNA template used for amplifying the qPCR standard by PCR shall be verified
by electrophoresis on 1 % agarose gel in TBE buffer stained with appropriate staining (e.g. SYBR Safe™
staining). The concentration of DNA is measured by spectro-photometry at 260 nm. DNA template is diluted
−1
to 10 ng·µl in a final volume of 20 µl and stored at −20 °C.
The qPCR standard sequence is amplified by PCR using a specific primer pair designed according to
recommendations described in task 1 of block 1. The amplification reaction is carried out in a final 25 µl
−1 −1
volume containing 2,5 µl of 10 × Taq polymerase buffer, 200 µmol·l of each dNTP, 1,5 mmol·l of MgCl ,
−1
0,5 µmol·l of each primer and 0,625 U of Taq polymerase. A volume of 2,5 µl of DNA (e.g. 25 ng of DNA)
is used as template for the PCR reactions. PCR is performed in a thermocycler according to the following
program: one cycle of 4 min at 94 °C; 39 cycles of 1 min at 94 °C, 1 min at annealing temperature specific for
the qPCR standard amplicon, 1,5 min at 72 °C, and a final extension step at 72 °C for 5 min. The expected size
of the qPCR standard amplicon is verified by electrophoresis on 2 % agarose gel in TBE buffer stained with
appropriate staining (e.g. SYBR Safe™ staining). Amplicons are purified either from the gel using appropriate
methods or by using exclusion chromatography columns to remove primers. Purified amplicons are then
quantified by spectro-photometry at 260 nm or by spectrofluorimetry.

oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
7.1.4.2 Cloning, dilution preparation of qPCR standard
7.1.4.2.1 Ligation of amplicon of qPCR standard
For an optimal ligation of an amplicon into a cloning vector should a 3:1 molar ratio the mass of PCR product
(Q in ng of DNA) to be used for ligation can be calculated (see Formula 1):
mn
plasmidDNA× insert
Q=×3 (1)
n
plasmid
Q = [(amount of plasmid DNA × size of the insert (bp)/size of the plasmid (bp)] × (3/1)
where
Q is the mass of PCR product, in nanograms (ng);
m is the mass of plasmid DNA, in nanograms (ng);
plasmid DNA
n is the size of the insert, in bp;
insert
n is the size of the plasmid, in bp;
plasmid
Taking into account that a plasmid size of 3 000 bp, a 16S rRNA insert of 200 bp, and 50 ng of plasmid DNA
per ligation reaction the amount of PCR amplicon to be used per ligation (see Formula 2) is:
50×200
Q= ×=310 (2)
The ligation reaction is made of the required amount of qPCR standard purified amplicon (Q), 50 ng of
plasmid DNA, 5 µl of 2 × ligation buffer, 3 U of T4 DNA ligase, and molecular grade water to reach a final
volume of 10 µl. The ligation reaction is incubated overnight at 4 °C or for adequate T4 DNA ligase, one hour
at ambient temperature.
The efficiency of the ligation is verified by electrophoresis by loading 1 µl ligated plasmid and open plasmid
(i.e. 5 ng of plasmid) on 1 % agarose gel in TBE buffer stained with appropriate staining. Ligated plasmid is
characterized by a shorter migration in the agarose gel.
7.1.4.2.2 Transformation of competent Escherichia coli
8 −1
Competent E. coli are transformed by heat shock as described below. Competent cells (10 cfu·µg of DNA)
freshly thawed out are incubated for 5 min on ice. Then 1 µl of the ligation reaction is added to cells, smoothly
mixed, and incubated for 20 min on ice. Bacterial cells are heat shock treated for 50 s incubation at 42 °C and
immediately placed on ice and incubated for 2 min. Then 950 µl of SOC medium are added and the bacterial
−1
cells are incubated at 37 °C under agitation at 150 min for 1 h. 100 µl bacterial cells aliquots are plated
onto LB/Amp/IPTG/X-Gal solid medium. Petri dishes are then incubated at 37 °C overnight.
7.1.4.2.3 Screening for recombinant clone
Plates are placed at 4 °C for several hours to accentuate coloration of bacterial colonies. White colonies are
picked, plated onto LB/Amp/IPTG/X-Gal solid medium, and incubated overnight at 37 °C. Several white
colonies were picked and put in 100 µl molecular grade water. PCR is carried out to confirm the presence of
the insert in the recombinant clone. The insert is amplified by PCR using SP6 (5’-ATT TAG GTG ACA CTA TAG
−3’) and T7 (5’-TAA TAC GAC TCA CTA TAG GG −3’) primers. The amplification reaction is carried out in a
−1 −1
final 25 µl volume containing 2,5 µl of 10 × Taq polymerase buffer, 200 µmol·l of each dNTP, 1,5 mmol·l of
−1
MgCl , 0,5 µmol·l of each primer and 0,625 U of Taq polymerase. A volume of 2,5 µl of bacterial suspension
is used as template for the PCR reactions. PCR is performed in a thermocycler according to the following
program: one cycle of 4 min at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at 55 °C, 1,5 min at 72 °C, and a final
extension step at 72 °C for 5 min. The size of the expected qPCR amplicon is verified by electrophoresis on
2 % agarose gel in TBE buffer stained with appropriate staining.

oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
7.1.4.2.4 Purification and linearization of recombinant plasmid
Recombinant clones, white in colour confirmed by PCR are inoculated to 10 ml LB/Amp liquid medium
−1
incubated at 37 °C under agitation (150 min ) overnight. Plasmid is purified from 2 ml cell suspension
using conventional mini-preparation. Plasmid DNA is then quantified by spectro-photometry at 260 nm and
aliquots are prepared and stored at −20 °C until use.
Plasmid is linearized with a restriction enzyme presenting a single restriction site in the sequence of the
plasmid. User shall make sure that the chosen restriction enzyme is not also cutting the insert. Digestion
of the plasmid is performed overnight at 37 °C in a final volume of 10 µl containing 250 ng of recombinant
plasmid, 0,5 U of restriction enzyme, 1 µl of 10 × restriction enzyme buffer, and molecular grade water.
The efficiency of the restriction of the plasmid is verified by electrophoresis on 1 % agarose gel. Linearized
plasmid is stored at −20 °C and is used as stock solution to prepare serial dilution of qPCR standard used to
calibrate qPCR assay.
The concentration in DNA of linearized plasmid is measured by spectrophotometry at 260 nm or by
spectrofluorimetry in order to determine the plasmid copies number. This operation can be facilitated
by using an online calculator such as oligo calc (http:// www .basic .northwestern .edu/ biotools/ oligocalc
.html). From this stock solution, an initial solution containing 0,5 × 10 copies of the qPCR standard per µl
is prepared in 100 µl of molecular grade water. Tenfold serial dilutions are then prepared to reach 0,5 × 10
copies of the plasmid per µl. Additional intermediary dilutions can also be prepared depending on the range
where copy numbers are expected.
7.1.5 Calibration of the qPCR (task 1, step 3)
7.1.5.1 General
The procedure used to generate the calibration curve and evaluate the efficiency of the qPCR assay is
described thereafter.
7.1.5.2 qPCR assay
8 1
qPCR calibration assay is performed on serial dilution of the cloned standard (ranging from 10 to 10
copies per µl) using a primer pair specifically targeting the gene of interest. The amplification reaction is
−1
carried out in a final 15 µl volume containing 2 µl of plasmid standard, 1 µmol·l of each primer, 7,5 µl of
2 × Taq master mix or 1,5 µl of 10 × Taq master mix containing a fluorescent DNA binding dye, dNTPs, MgCl ,
and Taq polymerase and molecular grade water. qPCR reaction is performed in a real-time thermocycler
according to the following program: one cycle of 15 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at annealing
temperature, 30 s at 72 °C, 30 s at 80 °C where the fluorescence is collected, and a final dissociation stage by
increasing the temperature from 80 °C to 95 °C. qPCR calibration is performed in triplicate and three NTC
are also included.
7.1.5.3 Establishment of the calibration curve and calculation of qPCR efficiency
At the end of qPCR assay, results are analyzed using the automatic option. Validation of the qPCR required
the observation of: a) no amplification for NTC, b) a single dissociation peak for each dilution of qPCR
standard, and c) a linear calibration curve with r equal or superior to 98 %. qPCR calibration curve is giving
the number of Ct as a function of the amount of the log of the number of copy of standard sequences.
The efficiency of the qPCR assay is estimated as given in Formula 3 from values determined from the
calibration curve formula.
Ct =⋅aq+c (3)
oSIST prEN ISO 17601:2024
ISO/DIS 17601:2024(en)
where
Ct is measured cycle threshold;
q is the copy number of qPCR standard;
a slope of the calibration curve;
c ordinate at the origin (Ct for 1 copy of qPCR standard).
()−1/a
E =−10 1 (4)
where
E is the efficiency of the calibration assay;
a slope of the calibration curve.
It shall be noticed that calibration curve having a slope equal to −3,32 is 100 % efficient. A twofold- or a
10-fold-dilution of a given DNA template gives a Ct difference of 1 or of 3,3 respectively (i.e. 10 Ct = 10 and
10 Ct = 13,3) for qPCR assay efficient at 100 %.
7.2 Preparation of soil DNA template and inhibition test (task 2)
7.2.1 General
This task describes the procedure used to prepare DNA template and to check for the presence of Taq
polymerase inhibitors in DNA extracts used as template for qPCR assay. This test is an obligatory prerequisite
to validate the quality of DNA extracts and allow their use as template for conducting qPCR assays.
7.2.2 Soil DNA preparation (task 2, step 4)
Step 4 of task 2 describes the procedure used to extract DNA from soil samples. Soil samples shall be
collected, handled, and stored according to ISO 10381-6. Soil DNA extraction shall be done according to
−1 −1
ISO 11063. Soil DNA samples are diluted to 1 ng·µl and 0,1 ng·µl and stored at −20 °C until their use.
7.2.3 Inhibition test (task 2, step 5)
The procedure used to test for the presence of Taq polymerase inhibitors in soil DNA extracts is described
thereafter. This step is prerequisite that shall be done prior to perform qPCR assay from soil DNA extracts.
Indeed, Taq polymerase inhibitors such as humic acid substances often co-extracted with soil DNA and only
soil DNA extract free of inhibitors can be subjected to qPCR analysis to estimate the abundance of selected
microbial gene sequences in soil. Two inhibition tests are described thereafter.
7.2.3.1 Spiking of exogenic DNA in soil DNA extract
Search for inhibitors can be done by quantifying the abundance of exogenic DNA spiked in known amount
to soil DNA. The protocol proposed below describes the analysis done after spiking plasmid DNA to soil
DNA extract. This procedure can be adapted to any exogenic DNA sources by performing the qPCR with
appropriate primer pair specific for the sequence of spiked DNA.
qPCR is performed using SP6 and T7 primers specific from the plasmid. The amplification reaction is carried
out in a final 15 µl volume containing 2 µl of plasmid DNA, 2 µl of soil DNA (using the two dilutions 1 and
−1 −1
0,1 ng·µl ), 1,0 µmol·l of each primer, 7,5 µl of 2 × Taq master mix or 1,5 µl of 10 × Taq master mix containing
fluorescent DNA binding dye dNTPs, MgCl , and Taq polymerase and molecular grade water. qPCR reaction
is performed in a real-time thermocycler according to the following program: one cycle of 15 min at 95 °C;
30 cycles of 15 s at 95 °C, 30 s at 55 °C, and 30 s at
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