SIST-TS CEN/TS 16707:2014
(Main)Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Polymerase chain reaction (PCR) based screening strategies
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Polymerase chain reaction (PCR) based screening strategies
This Technical Specification describes screening strategies for the detection of genetically modified (GM) DNA in food products by means of PCR methods. The strategies have been established for food matrices, but it can also be applied to other matrices (e.g. feed, seed and samples from field grown plants).
Detection of GM DNA is based on PCR methods targeting segments of transgenic DNA sequences (genetic elements, genetic constructs or insertion sites of transgenes). Various combinations of these PCR methods are involved in screening strategies. The methods are applied simultaneously or hierarchically. The general strategy is based on the matrix approach. Examples for the implementation and application of this approach are described.
In order to ensure reliable analytical results, the document also provides guidelines for the validation of the performance criteria of qualitative PCR methods applied in screening approaches.
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihren Produkten - Strategies für das Screening mit Polymerase-Kettenreaktion (PCR)
Diese Technische Spezifikation beschreibt Strategien für den Nachweis gentechnisch veränderter (gv-) DNA in Lebensmitteln mittels real-time PCR Verfahren. Sie wurde für Lebensmittelmatrices erarbeitet, kann jedoch auch auf andere Matrices angewendet werden (z. B. Futtermittel und aus der Umwelt entnommene Pflanzenproben).
Der PCR Nachweis von gv-DNA basiert auf dem Targeting von Abschnitten transgener DNA Sequenzen (genetischer Elemente, genetischer Konstrukte oder Insertionsstellen von Transgenen). Dieses Dokument beschreibt die Vorgehensweise zur effektiven Untersuchung einer Probe auf Anwesenheit von DNA, die auf gv-Material zurückgeht. Die Nachweisverfahren werden hierbei gleichzeitig oder hierarchisch, in nacheinander ablaufenden Reaktionen kombiniert. Die Untersuchung basiert auf dem sogenannten "Matrix approach" als generelle Analysestrategie. Es werden Beispiele für die Einführung und Anwendung dieser Vorgehensweise dargelegt.
Um verlässliche Ergebnisse auf diesem Untersuchungsgebiet zu gewährleisten, beschreibt das Dokument außerdem Leitlinien für die Validierung der Leistungskriterien von PCR-Verfahren, die beim Screening zum Einsatz kommen.
Produits alimentaires - Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés - Stratégies de criblage basées sur l'utilisation de la réaction de polymérisation en chaîne (PCR)
La présente Spécification technique décrit des stratégies de criblage pour la détection d’ADN génétiquement modifié (GM) dans les produits alimentaires au moyen des méthodes de PCR. Ces stratégies ont été élaborées pour les matrices alimentaires mais elles peuvent également être appliquées à d’autres matrices (par exemple aliments pour animaux, graines et échantillons issus de plantes cultivées en plein champ).
La détection d’ADN GM est basée sur des méthodes de PCR ciblant des segments de séquences d’ADN transgéniques (éléments génétiques, construits génétiques ou sites d’insertion de transgènes). Les stratégies de criblage font intervenir différentes combinaisons de ces méthodes de PCR. Ces méthodes sont appliquées simultanément ou de façon hiérarchique. La stratégie générale repose sur l’approche matricielle. Des exemples de mise en oeuvre et d’application de cette approche sont décrits.
Pour garantir des résultats analytiques fiables, le document fournit également des lignes directrices pour la validation des critères de performance des méthodes de PCR qualitative utilisées dans les approches de criblage.
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in njihovih produktov - Strategije presejalne analize s polimerazno verižno reakcijo (PCR)
Ta tehnična specifikacija opisuje strategije presejalne analize za odkrivanje gensko spremenjenega (GM) DNK-ja v živilskih proizvodih z metodo PCR. Te strategije so bile pripravljen za živilske matrice, vendar se lahko uporabljajo tudi za druge matrice (npr. za vzorce krme, semen in rastlin, gojenih na polju).
Odkrivanje GM DNK-ja temelji na metodah PCR za iskanje transgenih sekvenc DNK (genskih elementov, genske konstrukte ali mest vstavitve transgenov). V strategije presejalne analize so vključene različne kombinacije teh metod PCR. Metode se uporabljajo hkrati ali hierarhično. Splošna strategija temelji na pristopu matrice. Opisani so primeri implementacije in uporabe tega pristopa.
Za zagotovitev zanesljivih rezultatov analize podaja dokument tudi smernice za preverjanje meril učinkovitosti uporabljenih kvantitativnih metod PCR v pristopih presejalne analize.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 16707:2014
01-december-2014
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov - Strategije presejalne analize s polimerazno verižno reakcijo
(PCR)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Polymerase chain reaction (PCR) based screening strategies
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Strategies für das Screening mit Polymerase-Kettenreaktion (PCR)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes
génétiquement modifiés et des produits dérivés - Stratégies de criblage basées sur
l'utilisation de la réaction de polymérisation en chaîne (PCR)
Ta slovenski standard je istoveten z: CEN/TS 16707:2014
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST-TS CEN/TS 16707:2014 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 16707:2014
TECHNICAL SPECIFICATION
CEN/TS 16707
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
October 2014
ICS 67.050
English Version
Foodstuffs - Methods of analysis for the detection of genetically
modified organisms and derived products - Polymerase chain
reaction (PCR) based screening strategies
Produits alimentaires - Méthodes d'analyse pour la Lebensmittel - Verfahren zum Nachweis von gentechnisch
détection des organismes génétiquement modifiés et des veränderten Organismen und ihren Produkten - Strategien
produits dérivés - Stratégies de criblage basées sur für das Screening mit Polymerase-Kettenreaktion (PCR)
l'utilisation de la réaction de polymérisation en chaîne
(PCR)
This Technical Specification (CEN/TS) was approved by CEN on 28 June 2014 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
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, 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: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16707:2014 E
worldwide for CEN national Members.
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Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 Principle .6
5 Reagents .7
5.1 General .7
5.2 PCR reagents .7
6 Apparatus and equipment .7
7 PCR analysis .7
7.1 General .7
7.2 Screening .7
7.2.1 General .7
7.2.2 Combination of targets .8
7.2.3 Analysis of the output of the first screening .9
7.2.4 Additional screening tests .9
7.3 GM event identification .9
7.3.1 Event specific tests .9
7.3.2 Additional tests . 10
7.4 Interpretation of PCR results . 10
7.4.1 General . 10
7.4.2 Interpretation of results at the limit of detection (LOD) . 10
8 PCR method performance criteria and validation . 11
8.1 General . 11
8.2 Absolute limit of detection (LOD ) . 12
abs
8.3 Specificity and reference materials . 12
8.4 Robustness . 13
8.5 False-positive rate and false-negative rate . 13
8.6 Probability of Detection (POD) . 13
Bibliography . 14
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Foreword
This document (CEN/TS 16707:2014) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] 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 announce this Technical Specification: 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
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Introduction
Largely, detection of materials derived from genetically modified organisms (GMOs) in a given sample
employs polymerase chain reaction (PCR) analysis, specifically real-time PCR.
A general strategy for GMO detection and identification by means of PCR analysis and a stepwise approach is
described.
In initial screening analysis, DNA sequences of genetic elements common to many GMOs are targeted.
According to its purpose, screening is a test to rapidly and reliably sort samples into groups. Once the
samples are grouped, screening facilitates and potentially reduces subsequent analytical work and results
interpretation. The screening strategy should be adjusted to the scope (food, feed or seed, crop-specific etc.)
of the test(s).
This document takes the general principle of GMO detection strategies as a basis and describes the
underlying analytical steps for complex screening (known as the matrix-approach [2]).
The document is written primarily for screening strategies applying real-time PCR methods. Other PCR
methodologies may be applicable in the same way.
The terms “screening method” and “screening strategies” are not interchangeable and have different
meanings in this document.
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1 Scope
This Technical Specification describes screening strategies for the detection of genetically modified (GM) DNA
in food products by means of PCR methods. The strategies have been established for food matrices, but it
can also be applied to other matrices (e.g. feed, seed and samples from field grown plants).
Detection of GM DNA is based on PCR methods targeting segments of transgenic DNA sequences (genetic
elements, genetic constructs or insertion sites of transgenes). Various combinations of these PCR methods
are involved in screening strategies. The methods are applied simultaneously or hierarchically. The general
strategy is based on the matrix approach. Examples for the implementation and application of this approach
are described.
In order to ensure reliable analytical results, the document also provides guidelines for the validation of the
performance of qualitative PCR methods applied in screening approaches.
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.
EN ISO 21569, Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Qualitative nucleic acid based methods (ISO 21569)
EN ISO 21570, Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Quantitative nucleic acid based methods (ISO 21570)
EN ISO 21571, Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Nucleic acid extraction (ISO 21571)
EN ISO 24276, Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - General requirements and definitions (ISO 24276)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN ISO 24276 and the following apply:
3.1
GMO method matrix
relational presentation (e.g. a table) of symbols or numbers
Note 1 to entry: One dimension (e.g. columns) corresponds to genetic elements and genetic constructs detected by a
defined PCR method and the other dimension (e.g. rows) corresponds to GM events. The entered symbols or numbers
indicate the detectability or non-detectability of the target sequence for the GM event.
Note 2 to entry: The term matrix is commonly used for a defined composition of food, but this definition is not relevant
here.
3.2
GMO target matrix
relational presentation (e.g. a table) of symbols or numbers
Note 1 to entry: One dimension corresponds to genetic elements or genetic constructs present in a GMO and the
other dimension (e.g. rows) corresponds to GM events. The entered symbols or numbers indicate the presence or
absence of the target for the GM event and copy number, if available.
Note 2 to entry: In contrast to GMO method matrix, the GMO target matrix is independent from a detection method.
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3.3
screening method
method that rapidly and reliably eliminates (screens) a large number of negative (or positive) test samples and
restricts the number of test samples requiring the application of a rigorous method
Note 1 to entry: In this document, a screening method refers to PCR methods detecting the presence of several
GMOs in one test.
3.4
element-specific method
method that targets a single discrete DNA sequence of a specific genetic element
Note 1 to entry: A genetic element is a part of a gene, for example a promoter, terminator, intron or a coding
sequence. Elements are often derived from naturally occurring viruses, bacteria, plants, etc. However, elements in GMOs
are commonly modified at the sequence level, relative to the original (natural) source, e.g. by altered codon-usage or
specific single nucleotide polymorphisms (SNPs). This sequence modification may be based on adaptation of the
nucleotide composition to the new host genome.
3.5
construct specific method
method that targets a combination of inserted DNA sequences that are only found in GMO-derived material
composed of at least two elements that do not naturally co-exist in this conformation, and where the 5’ and 3’
end of the sequence are derived from separate genetic elements
3.6
event specific method
method that detects a specific sequence that is only present in that event
Note 1 to entry: The event may be the result of either a rearrangement or unique combination of insert and insertion
locus during the transformation.
Note 2 to entry: This is commonly targeted at the integration-border region.
3.7
probability of detection (POD)
probability of a positive analytical outcome for a qualitative method for a given matrix at a given concentration
Note 1 to entry: It is estimated by the expected proportion of positive results for the given matrix at the given analyte
concentration.
4 Principle
Total DNA is extracted from the sample by a suitable extraction method. The DNA quantity and quality shall
be checked as specified in EN ISO 21571 to ensure the presence of sufficient analyte and to assess the
presence of PCR inhibitors that could inhibit PCR amplification.
Subsequently, a set of validated PCR methods are selected and applied in a decision tree approach, for
detection and identification of genetic modifications linked to the sample. This approach enables the user to
choose between different courses of action. Commonly, screening starts with element-specific PCR tests prior
to construct-specific and/or event-specific tests. Before, in parallel or after screening, taxon-specific tests are
recommended.
Based on the combination of results at each level of the decision tree the total number of tests required for the
analysis is reduced.
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5 Reagents
5.1 General
The requirements and conditions for nucleic acid extraction and PCR analysis laid down in detail in
EN ISO 24276, EN ISO 21569, EN ISO 21570 and EN ISO 21571 shall be followed. Only analytical grade
reagents suitable for molecular biology shall be used.
5.2 PCR reagents
1)
5.2.1 Concentrated PCR buffer solution (containing reaction buffer, dNTPs, MgCl and Hotstart Taq
2
polymerase) or equivalent.
5.2.2 Oligonucleotides at the concentrations specified in the method protocol.
6 Apparatus and equipment
See EN ISO 24276, EN ISO 21569, EN ISO 21570 and EN ISO 21571.
7 PCR analysis
7.1 General
A large number of validated PCR methods applicable for the detection of GM DNA in food samples are
available in EN ISO 21569, EN ISO 21570 or are compiled in [3] and [4]. The complexity of these methods
can be divided in four categories according to the targeted DNA sequence:
— taxon-specific (for the detection and identification of DNA of a species or a taxonomical group);
— element-specific (for the detection and identification of genetic elements present in more than one GMO);
— construct-specific (for the detection and identification of specific combinations of genetic elements
present in a GMO as a result of genetic engineering);
— event-specific (for the detection and identification of the insertion site specific for a GM event).
Testing laboratories develop a strategy for selecting the methods according to their scope (food, feed or seed
testing), and requirements of their clients (e.g. government authority or company). In addition, the incidence of
products on the market containing or consisting of new GMOs may require the ad hoc implementation of new
methods into an existing screening strategy in order to incorporate the detection and identification of the
respective GM target DNA.
To confirm the identity of the PCR product generated by the PCR method, the method shall include a step for
verification of the amplicon by an appropriate technique (e.g. probe hybridization, subsequent DNA sequence
analysis or restriction enzyme digestion of the PCR product, respectively).
7.2 Screening
7.2.1 General
The first step of PCR based screening analysis is the selection of a defined set of methods targeting DNA-
sequences present in several GM events. The set of methods selected depends on the availability of
(validated) methods, the capability of the laboratory and the scope of the testing.
1) Ready-to-use reagent mixtures or single components may be used as PCR buffer solution. If other reagent mixtures
are used than the ones stated in the method validation report, they should give comparable to or better results.
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The selection of methods should allow a maximum of information obtained from a minimum number of the
tests performed in the first step. The selection of methods should also allow a maximum coverage of the
GM events under scrutiny, when applicable.
Selected methods should be evaluated for their sensitivity (8.2) and specificity (8.3).
7.2.2 Combination of targets
Different combinations of targets can be chosen depending on the purpose of the analysis.
If genetic elements or constructs commonly present in GMOs are targeted, a ‘GMO target matrix’ or a ‘GMO
method matrix’ can be established for the interpretation of the combined outcome of these tests (Table 1).
Practical examples of application of a GMO method matrix are described [5], [6], [7], [9] and [10], an example
for an available GMO target matrix is given in Reference [7].
Table 1 is an example of a GMO method matrix used in screening for various GM events. The matrix is based
on experimental or theoretical (in silico) verifications. The example tabulates five screening methods
(“ScreenMeth” A to E) and seven GM events. Each GM event has a distinct profile of positive (denoted by a
‘+’ representing ‘target detected’) and negative (denoted by a ‘-‘ representing ‘target not detected’) reactions
with the set of screening methods [2]. The outcome of the PCR tests performed with the five methods allows
considering the presence of the GM event(s) in the sample. For instance, if positive results are obtained with
method A and B and negative results with methods C, D and E it can be assumed that “GM event 2” is
present in the sample, and not the other GM events.
The use of a ‘GMO method matrix’ should indicate the copy number of the target sequence in the GM event
and the absolute LOD (see 8.2) of each screening method, if available. Otherwise, it could lead to erroneous
conclusions concerning the GMO(s) actually present in the sample. For example, a negative result by method
A and a positive result by method B may be a consequence of (i) the disproportional copy number of the
target sequence present in the GM event and/or of (ii) significantly different absolute LODs of both methods.
Table 1 — Example of a GMO method matrix
Analytical method
GM event ScreenMeth A ScreenMeth B ScreenMeth C ScreenMeth D ScreenMeth E
GM event 1 + + + - -
GM event 2 + + - - -
GM event 3 + + - + -
GM event 4 + - - + -
GM event 5 - + + - -
GM event 6 - - - - +
GM event 7 + + - - +
The numbers of GMOs and PCR methods included in the GMO method matrix are flexible. Each variable can
be increased or decreased according to the (1) available information, (2) needs for the specific analysis and
(3) requested coverage. For the efficient use of GMO method matrices it is important to distinguish which
species DNAs are present, in order to discriminate and reduce the number of required tests. For example, if
the food sample consists of only a single species, the design and subsequent use of the GMO method matrix
can be filtered for the GMOs belonging to this species group.
Alternatively, the coverage of GMOs that are potentially detected in the first screening step can be categorized
based on the origin of the product (region, country, continent etc.) and available information on the use of
GMOs to that geographic region.
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Multiplex PCR methods (or other developed analysis platforms) are available for selected combinations of
screening targets for example in [8], [11] and [15].
If methods have significantly different absolute LODs (e.g. > 10 copies), they should not be combined in the
same GMO method matrix.
NOTE 1 According to the selected methods, the outcome may lead to a group of GM events instead of a single one.
NOTE 2 If different amounts of diverse target-species DNA in composite food products are determined, this may lead
to disparate practical LODs for the applied tests. Assorted absolute LODs of the detection methods also need to be
considered as this may also lead to dissimilar practical LODs.
7.2.3 Analysis of the output of the first screening
The GMO method matrix can be used to design decision trees. The pattern of the output (detection of specific
screening targets) indicates the presence or absence of one or several GM events listed in a method matrix.
The information obtained in a first round of screening can then be used to decide, which targets should be
selected for a second round of screening.
The target taxon(s) of the DNA under study shall be identified. The determination of the species present in the
sample could be done prior to, in parallel with, or after the first screening phase. If relevant, further
quantification shall be undertaken in order to assess the practical LOD.
After the first round of screening, the next step in the decision tree process is to consider the following:
— If the sample is negative, no further analysis is required;
— If the sample is positive,
— and the number of suspected events is low, event specific tests or tests for identification of the
event(s) should be performed,
— and the number of suspected events is high, a second round of screening may be required according
to the species determined.
For output analysis, it shall be considered that more than one GMO or stacked GM events could be present,
as well as a GMO(s) not listed in the GMO method matrix.
7.2.4 Additional screening tests
Further screening may be useful, where the output of the first screening round enables the sorting of GMOs
into groups of events distinguishable by presence/absence of another target (e.g. a specific construct of
genetic elements specific for a group of GM events). This additional screening test(s) could reduce the
number of GM event-specific tests. For example, decision tree tools are described for GMO detection in plant
products [4], [5], [6], [7], [8], [13] and [14].
7.3 GM event identification
7.3.1 Event specific tests
Neither interpretation nor conclusion about the presence of an event shall be made without stating the event-
specific method used.
It may not be possible after the initial screening analysis to determine all the GM events potentially present in
the sample by additional screening tests. If this is the case, the next step in the analysis is to set up tests for
GM event identification.
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Event-specific PCR methods are used to identify the GM event(s) unambiguously.
Different platforms can be used for GMO identification, either as individual event-specific tests or as groups of
events in (i) prepared multi-well PCR strips or plates [8], [12], or (ii) multiplex PCR assays [11], [15]. The use
of multi-well strips or plates may be also an applicable strategy for direct analysis of a sample for GMO
identification. In some events, a genetic element or construct could be present in more than one copy per
haploid genome. Therefore, if the GM DNA content is low, it is possible to obtain a positive result in the
screening test, but a negative one in the event-specific test that is by definition a single copy target sequence.
NOTE Occasionally a unique genetic construct is present in a single GM event or a certain group of GM events.
Assignment of a GMO to the corresponding group can then be achieved also by an appropriate construct-specific method,
although the specific identity of the GMO cannot be determined unambiguously.
7.3.2 Additional tests
The output from the GMO identification step may not completely explain the results of the screening. Thus,
additional tests can be performed to reduce the room for interpretation:
— It is recommended to first sum up the output of the taxon-specific tests. Then, consider the presence of
other species than those indicated (or apparently present in the sample material). Traces of material from
other species than those indicated could be caused by adventitious and technically unavoidable
admixture during primary production (harvest, transport, storage). In addition, the contact to GMO-derived
packaging materials (starch, cotton, flax, etc.), or the content of spices should be considered.
— The copy numbers of the targeted elements could be disproportionately present in some GMOs (see
7.2.2). This condition needs to be considered for the decision of further required testing.
— If a positive result is obtained with a method targeting a single genetic element and this signal cannot be
related to the presence of a GMO, it is useful to test the sample for the presence of DNA derived from the
donor organism (e.g. testing for CaMV in case of a positive P-35S signal not explained by the GMO
identification step).
Some widely used GMOs occur frequently and their presence should be considered in some food products.
NOTE PCR methods are available to rule out the possibility that the detected signal is due to the presence of DNA
derived from naturally occurring organisms [16], [17] and [18].
7.4 Interpretation of PCR results
7.4.1 General
Results from all test portions of a sample shall be consistent. When at least one test portion gives a positive
result and at least one gives a negative result, the analysis shall be repeated [see EN ISO 24276].
NOTE 1 If possible, the quantity of template DNA in the reaction can be increased, in order to get consistent results for
both test portions.
NOTE 2 Verification of a positive detection is obtained e.g. by sequence-specific hybridization of the real-time
hydrolysis probe.
7.4.2 Interpretation of results at the limit of detection (LOD)
For the interpretation of results obtained with the matrix approach using screening tests it should be
considered that the practical LOD depends on (i) the method performance, (ii) the quantity of the target taxon
DNA and (iii) the number of target sequence copies per haploid genome in the GM event(s) present in the
sample [EN ISO 24276]. The copy number of individual targets
...
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