SIST EN ISO 17601:2026

SLOVENSKI STANDARD
01-april-2026
Nadomešča:
SIST EN ISO 17601:2018
Kakovost tal - Ocena številčnosti izbranih sekvenc mikrobnih genov s
kvantitativno verižno reakcijo s polimerazo (qPCR) v talnih ekstraktih DNK (ISO
17601:2025)
Soil quality - Estimation of abundance of selected microbial gene sequences by
quantitative polymerase chain reaction (qPCR) from DNA directly extracted from soil
(ISO 17601:2025)
Bodenbeschaffenheit - Ermittlung der Häufigkeit ausgewählter mikrobieller
Gensequenzen durch quantitative PCR aus DNA-Boden-Extrakten (ISO 17601:2025)
Qualité du sol - Estimation de l’abondance de séquences de gènes microbiens par
amplification par réaction de polymérisation en chaîne quantitative (qPCR) à partir
d’ADN directement extrait du sol (ISO 17601:2025)
Ta slovenski standard je istoveten z: EN ISO 17601:2025
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
November 2025
EUROPÄISCHE NORM
ICS 13.080.30 Supersedes EN ISO 17601:2018
English Version
Soil quality - Estimation of abundance of selected
microbial gene sequences by quantitative polymerase
chain reaction (qPCR) from DNA directly extracted from
soil (ISO 17601:2025)
Qualité du sol - Estimation de l'abondance de Bodenbeschaffenheit - Ermittlung der Häufigkeit
séquences de gènes microbiens par amplification par ausgewählter mikrobieller Gensequenzen durch
réaction de polymérisation en chaîne quantitative quantitative PCR aus DNA-Boden-Extrakten (ISO
(qPCR) à partir d'ADN directement extrait du sol (ISO 17601:2025)
17601:2025)
This European Standard was approved by CEN on 3 November 2025.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye 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
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17601:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 17601:2025) has been prepared by Technical Committee ISO/TC 190 "Soil
quality" in collaboration with Technical Committee CEN/TC 444 “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 May 2026, and conflicting national standards shall be
withdrawn at the latest by May 2026.
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.
This document supersedes EN ISO 17601:2018.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 17601:2025 has been approved by CEN as EN ISO 17601:2025 without any modification.

International
Standard
ISO 17601
Second edition
Soil quality — Estimation of
2025-10
abundance of selected microbial
gene sequences by quantitative
polymerase chain reaction (qPCR)
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 quantitative (qPCR) à partir d’ADN
directement extrait du sol
Reference number
ISO 17601:2025(en) © ISO 2025
ISO 17601:2025(en)
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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 intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 17601:2025(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 Enzymes .4
5.5 Chemicals .4
5.6 Products for bacterial culture medium .5
5.7 Buffers 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 Bacterial 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
7.3 qPCR assay (task 3) . . . 12
7.3.1 General . 12
7.3.2 qPCR (task 3, step 6) . 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 (task 4, step 7) . 12
7.4.3 Calculation of the copy number of the gene of interest in the soil DNA extract
(task 4, step 8) . 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 17601:2025(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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4, Biological
characterization, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 444, Environmental characterization of solid matrices, in accordance with the Agreement
on technical cooperation between ISO and CEN (Vienna Agreement).
This second edition cancels and replaces the first edition (ISO 17601:2016), which has been technically
revised.
The main changes are as follows:
— Annex C has been expanded by adding examples of well-established qPCR systems to quantify certain
microbial groups or their function.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO 17601:2025(en)
Introduction
DNA (DNAs) is a major component of any living organism, 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 eukaryotes).
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 the
[2][3][4][5][6]
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 analyses of soil microbial communities and 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 method
[7][4][8][9]
chosen for the analysis.
Numerous studies have investigated new methods to improve extraction, purification, amplification,
[10]
and quantification of DNA from soils. Recently, ISO 11063 reported “a method to extract nucleic acids
directly from soil samples” derived from Reference [10], opening a new window for developing standardized
[11]
molecular approaches to estimate soil quality.
The aim of this document 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 and functional groups by qPCR assays can contribute to
the development of routine tools to monitor soil quality. The repeatability and the reproducibility of the qPCR
procedure were assessed in an international ring test study (see Annex B). The repeatability of this procedure
was successfully evaluated for 16S rRNA genes and for 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. 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.
v
International Standard ISO 17601:2025(en)
Soil quality — Estimation of abundance of selected microbial
gene sequences by quantitative polymerase chain reaction
(qPCR) from DNA directly extracted from soil
1 Scope
This document specifies the crucial steps of a quantitative polymerase chain reaction (qPCR) method to
measure the abundance of selected microbial gene sequences from soil DNA extract. The number of microbial
gene sequences quantified by qPCR provides an estimation of the abundance of selected microbial groups in soil.
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 18400-206, Soil quality — Sampling — Part 206: 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 living and dead biota in the soil
Note 1 to entry: The soil biota includes microorganisms, plants and animals.
3.2
polymerase chain reaction
PCR
method allowing the amplification of a specific DNA sequence using a specific pair of oligonucleotide primers
3.3
quantitative polymerase chain reaction
qPCR
method allowing the quantification in a DNA template (3.4) of the number of DNA sequences targeted by a
specific pair of oligonucleotide primers
3.4
template
DNA sample used to perform polymerase chain reaction (PCR) (3.2) to amplify a specific DNA sequence

ISO 17601:2025(en)
3.5
amplicon
PCR product obtained by polymerase chain reaction (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 and which is used to transform
competent Escherichia coli
3.7
qPCR standard
cloned DNA target used as template (3.4) for quantitative polymerase reaction (qPCR) (3.3) to establish the
standard curve relating the abundance of target sequence as a function of cycle threshold (Ct) (3.9) values
3.8
non-template control
NTC
control, usually molecular grade water, that is used as negative control in quantitative polymerase reaction
(qPCR) (3.3) assay to check for the absence of contaminant in the qPCR mix
3.9
cycle threshold
Ct
number of quantitative polymerase chain reaction (qPCR) (3.3) cycles required for the fluorescent signal to
cross the threshold (i.e. to exceed background level)
Note 1 to entry: The Ct value is inversely proportional to the abundance of the target sequence.
4 Principle
This document describes a qPCR assay using fluorescent DNA binding dye as reporter. This qPCR assay has
1)
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 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.
1) SYBR® Green is a trademark of Thermo Fisher Scientific 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.

ISO 17601:2025(en)
Figure 1 — Main tasks and critical steps to estimate the abundance of selected microbial gene
sequences by qPCR assay
ISO 17601:2025(en)
This document describes a qPCR assay based on the use of fluorescent DNA binding dye which has been
®2)
validated by an international ring test using SYBR Green qPCR. In Annex A, information about the TaqMan
qPCR assay (which was not tested in the international ring test) is given. The first task comprises three steps
describing the design of optimal amplicon for qPCR (step 1), the preparation of qPCR standards (step 2),
and the procedure to calibrate the qPCR assay (step 3). The second task includes two steps describing the
procedures to prepare soil DNA samples (step 4) and to test for the presence of qPCR inhibitors in soil DNA
samples (step 5). The third task is constituted of a single step describing the protocol to perform the qPCR
assay (step 6).
Finally, the fourth task is made of two steps: one describes the procedure to validate the qPCR assays
(step 7) to check its quality and the other describes the different options to calculate the copy number of
gene sequences from the cycle threshold values (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 Enzymes
5.4.1 Taq polymerase.
5.4.2 T4 DNA ligase.
5.4.3 T4 gene 32 protein (T4gp32).
3)
5.4.4 Bovine serum albumin (CAS RN 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
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. ®
3) Chemical Abstract Service (CAS) Registry Number is a trademark of the American Chemical Society (ACS). 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.

ISO 17601:2025(en)
5.5.3 Deoxynucleotide solution, dNTPs.
®5)
5.5.4 SYBR Safe double-strand DNA gel stain.
5.5.5 Ethylenediaminetetraacetic acid disodium salt (EDTA) dihydrate, C H N O Na ·2 H O
10 14 2 8 2 2
(CAS No. 6381-92 6).
5.5.6 Glucose, C H O (CAS No. 50-99-7).
6 12 6
5.5.7 Hydrochloric 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 Products for bacterial culture medium
6)
5.6.1 Bacto™tryptone , enzymatic digest of casein.
5.6.2 Yeast extract powder (CAS No. 8013-01-2).
5.7 Buffers and reagents
5.7.1 Ampicillin 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 10 000 × 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
−1
agar in 1 000 ml of H O. After autoclaving for 20 min at 120 °C, 1 ml of ampicillin stock solution at 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-ampicillin medium. When IPTG solution is entered in LB-ampicillin medium,
20 µl of X-Gal solution is plated on solid LB-ampicillin medium. Solid LB medium is stored at 4 °C until its use.

ISO 17601:2025(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
5.7.8 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.
5.7.9 TBE buffer × 1, 100 ml of TBE buffer × 10 in 900 ml of H O.
−1 −1
5.7.10 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.
5.7.11 TE buffer × 1, 100 ml of TE buffer × 10 in 900 ml of H O.
5.7.12 X-gal solution, 250 mg of X-Gal in 5 ml of dimethylformamide. 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 Thermocycler suitable for qPCR analysis, 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
analysed.
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
The 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 the appropriate amplicon to settle down a qPCR assay (step 1), the preparation of
the qPCR standard (step 2), and the calibration of the qPCR assay (step 3).
7.1.2 Amplicon design (task 1, step 1)
7.1.2.1 General
The first step aims at designing the 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

ISO 17601:2025(en)
4)
available in the Genbank database . Only primers specific for the gene target shall be considered. 7.1.2.2
provides indications to guide the design of the oligonucleotide primer pair for establishing the qPCR assay.
7.1.2.2 qPCR
— Optimal amplicon length ranges between 100 bp (base pairs) 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 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 harbouring the qPCR standard for further use
for qPCR assays.
7.1.4 Bacterial isolate DNA, environmental DNA, artificial DNA
7.1.4.1 General
The first step of the 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:
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 spectrophotometry 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 the
recommendations described in 7.1.2.2 (task 1, step 1). The amplification reaction is carried out in a final
−1 −1
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 DNA (e.g. 25 ng
of DNA) is used as template for the PCR reactions. PCR is performed in a thermocycler according to the
recommendations of the qPCR kit furnisher. As an example, the following program can be used: 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, and 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
4) http:// www .ncbi .nlm .nih .gov/ genbank/

ISO 17601:2025(en)
by using exclusion chromatography columns to remove primers. Purified amplicons are then quantified by
spectrophotometry at 260 nm or by spectrofluorimetry.
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, a 3:1 molar ratio should be used. The mass of
PCR product to be used for ligation can be calculated using Formula (1):
mn
plasmidDNA× insert
Q=×3 (1)
n
plasmid
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 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 calculated using Formula (1) is:
50×200
Q= ×=310
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 by 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
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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 (5.7.5). 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 are 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, and 1,5 min at 72 °C; and a final

ISO 17601:2025(en)
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.
7.1.4.2.4 Purification and linearization of recombinant plasmid
Recombinant clones, white in colour and confirmed by PCR, are inoculated to 10 ml LB/Amp liquid medium
−1
incubated at 37 °C under agitation (150 min ) overnight. The plasmid is purified from 2 ml cell suspension
using conventional mini-preparation. Plasmid DNA is then quantified by spectrophotometry at 260 nm and
aliquots are prepared and stored at −20 °C until use.
The 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
5)
by using an online calculator such as oligo calc . 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 in 7.1.5.2 and 7.1.5.3.
7.1.5.2 qPCR assay
8 1
The 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
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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. The qPCR reaction is performed in a real-time thermocycler
according to the recommendations of the qPCR kit furnisher. As an example, the following program can be
used: 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, and
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. The temperature to which the fluorescence is measured shall be lower than the melting
temperature of the amplicon. 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 analysed using the program furnished with the thermocycler. In order
to validate the qPCR, the following shall be observed:
a) no amplification for NTC;
b) a single dissociation peak for each dilution of qPCR standard;
c) a linear calibration curve with r equal or superior to 98 %.
The qPCR calibration curve gives the number of Ct as a function of the amount of the log of the number of
copy of standard se
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