SIST 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 - Abschätzung 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
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-julij-2018
.DNRYRVWWDO2FHQDãWHYLOþQRVWLL]EUDQLKVHNYHQFPLNUREQLKJHQRYV
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
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
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, 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.
iv © ISO 2016 – All rights reserved
ISO 17601:2016(E)
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
[2]
providing unique insight into the composition, richness, and structure of microbial communities.
[3] [4] [5] [6]
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
[7] [4] [8] [9]
concentration of amplified DNA, errors in the PCR, or even the method chosen for analysis.
Numerous studies have investigated new methods to improve extraction, purification, amplification,
[10]
and 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
[11]
standardized 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.
INTERNATIONAL STANDARD ISO 17601:2016(E)
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.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 10381-6, 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 — Method to directly extract DNA from soil samples
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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
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
ISO 17601:2016(E)
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.
2 © ISO 2016 – All rights reserved
ISO 17601:2016(E)
Validation
Validation
Figure 1 — Main tasks and critical steps to estimate the abundance of selected microbial gene
sequences by qPCR assay
This International Standard describes qPCR assay based on the use of fluorescent DNA binding dye
®1)
which has been validated by an international ring test using SYBR Green qPCR. In Annex A,
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.
ISO 17601:2016(E)
2) ®
information about 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. ®
5.5.4 SYBR Safe DNA gel stain.
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.
4 © ISO 2016 – All rights reserved
ISO 17601:2016(E)
5.5.5 Ethylenediaminetetraacetic acid disodium salt (EDTA), 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 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.
ISO 17601:2016(E) ®
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
4 2
aliquots 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.
6 © ISO 2016 – All rights reserved
ISO 17601:2016(E)
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
-1
diluted 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
-1 -1
25 µl volume containing 2,5 µl of 10 × Taq polymerase buffer, 200 µmol·l of each dNTP, 1,5 mmol·l
-1
of 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 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.
ISO 17601:2016(E)
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
plasmid DNAi× nsert
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
-1
medium are added and the bacterial 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
-1
is carried out in a final 25 µl volume containing 2,5 µl of 10 × Taq polymerase buffer, 200 µmol·l of each
-1 -1
dNTP, 1,5 mmol·l of 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,
8 © ISO 2016 – All rights reserved
ISO 17601:2016(E)
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.
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
-1
is 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.
ISO 17601:2016(E)
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)
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.
10 © ISO 2016 – All rights reserved
SIST EN I
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.
Loading comments...