CEN/TS 17329-2:2019
(Main)Foodstuffs - General guidelines for the validation of qualitative real-time PCR methods - Part 2: Collaborative study
Foodstuffs - General guidelines for the validation of qualitative real-time PCR methods - Part 2: Collaborative study
This document provides information on how the performance characteristics of qualitative (binary) real-time polymerase chain reaction (PCR) methods for detection of specific DNA sequences present in foods should be evaluated and validated by conducting a collaborative study.
The guidelines are applicable for validation of qualitative PCR methods used for detection of DNA sequences derived from genetically modified foodstuffs. They can be applicable also for PCR methods used for detection of other target sequences in foodstuffs, e.g. for species detection and identification.
Lebensmittel - Allgemeine Anleitung für die Validierung qualitativer Realtime-PCR-Verfahren - Teil 2: Ringversuch
Dieses Dokument legt fest, wie die Leistungsmerkmale von Verfahren der qualitativen (binären) Realtime-Polymerasekettenreaktion (PCR) zum Nachweis von bestimmten, in Lebensmitteln vorliegenden DNA-Sequenzen in einem Ringversuch bewertet und validiert werden sollten.
Der Leitfaden wurde entwickelt für qualitative PCR-Verfahren zum Nachweis von DNA-Sequenzen, die von gentechnisch modifizierten Lebensmitteln stammen. Darüber hinaus lässt er sich anwenden bei PCR-Verfahren zum Nachweis von anderen Arten von Zielsequenzen in Lebensmitteln, z. B. für den Nachweis und die Identifizierung von Spezies.
Denrées alimentaires - Lignes directrices générales pour la validation des méthodes de PCR qualitative en temps réel - Partie 2 : Étude interlaboratoires
Le présent document fournit des informations sur la façon dont il convient d’évaluer et de valider, en effectuant une étude interlaboratoires, les caractéristiques de performance des méthodes de réaction en chaîne par polymérase (PCR) qualitative (binaire) en temps réel applicables à la détection de séquences d’ADN spécifiques présentes dans les aliments.
Les lignes directrices sont applicables à la validation des méthodes de PCR qualitative utilisées pour la détection de séquences d’ADN extraites de produits alimentaires génétiquement modifiés. Elles peuvent également être applicables aux méthodes de PCR utilisées pour la détection d’autres séquences cibles dans les produits alimentaires, par exemple pour la détection et l’identification des espèces.
Živila - Splošne smernice za validacijo kvalitativnih metod PCR v realnem času - 2. del: Medlaboratorijska študija
Ta dokument vsebuje informacije o tem, kako naj bi z izvedbo laboratorijske validacijske študije za kvalitativne (binarne) metode polimerazne verižne reakcije (PCR) v realnem času, ki se uporabljajo za odkrivanje določenih zaporedij DNA, prisotnih v živilih, ovrednotili in potrdili lastnosti delovanja.
Smernice se uporabljajo za validacijo kvalitativnih metod PCR za odkrivanje zaporedij DNA, pridobljenih iz gensko spremenjenih živil. Mogoče jih je uporabiti tudi za metode PCR, ki se uporabljajo za odkrivanje drugih ciljnih zaporedij v živilih, npr. za odkrivanje in identifikacijo vrst.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-julij-2019
Živila - Splošne smernice za validacijo kvalitativnih metod PCR v realnem času - 2.
del: Medlaboratorijska študija
Foodstuffs - General guidelines for the validation of qualitative real-time PCR methods -
Part 2: Collaborative study
Lebensmittel - Allgemeine Anleitung für die Validierung qualitativer Realtime-PCR-
Verfahren - Teil 2: Ringversuch
Denrées alimentaires - Lignes directrices générales pour la validation des méthodes de
PCR qualitative en temps réel - Partie 2 : Étude interlaboratoires
Ta slovenski standard je istoveten z: CEN/TS 17329-2:2019
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
CEN/TS 17329-2
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
April 2019
TECHNISCHE SPEZIFIKATION
ICS 67.050
English Version
Foodstuffs - General guidelines for the validation of
qualitative real-time PCR methods - Part 2: Collaborative
study
Denrées alimentaires - Lignes directrices générales Lebensmittel - Allgemeine Anleitung für die
pour la validation des méthodes de PCR qualitative en Validierung qualitativer Realtime-PCR-Verfahren - Teil
temps réel - Partie 2 : Étude interlaboratoires 2: Ringversuch
This Technical Specification (CEN/TS) was approved by CEN on 25 February 2019 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, 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17329-2:2019 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Validation of the performance characteristics by means of a collaborative study . 6
6 Calculation of precision data for test samples . 11
7 Study report . 11
Annex A (informative) Instructions for the conduct of the collaborative study . 12
Annex B (informative) Statistical model . 20
Bibliography . 23
European foreword
This document (CEN/TS 17329-2:2019) 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 shall not be held responsible for identifying any or all such patent rights.
This Technical Specification consists of two parts:
— Part 1: Single-laboratory validation
— Part 2: Collaborative study
According to the CEN/CENELEC Internal Regulations, the national standards organisations 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Introduction
Qualitative real-time polymerase chain reaction (PCR) methods currently find broad application for the
detection of specific DNA sequences in food, e.g. for the detection and identification of genetically
modified organisms and the products derived thereof, for food authentication and speciation and other
purposes. It is important that results obtained from different laboratories by such food analytical
methods satisfy certain performance characteristics and quality criteria. The performance of a method
is validated in a step-wise process from in-house (single laboratory) validation to a pre-validation study
by few laboratories followed by a full validation in a collaborative study to gain information and data on
the reproducibility of the analysis results obtained by different laboratories.
The aim of this document is to provide practical guidance for a collaborative validation study of
qualitative real-time PCR methods which are applied for food analysis. The procedure described is a
recommendation that is underpinned by practical experience in several collaborative trial studies. It is
possible to apply alternative approaches for which it can be shown that the performance criteria
mentioned in the present document are achieved.
1 Scope
This document provides information on how the performance characteristics of qualitative (binary)
real-time polymerase chain reaction (PCR) methods for detection of specific DNA sequences present in
foods should be evaluated and validated by conducting a collaborative study.
The guidelines are applicable for validation of qualitative PCR methods used for detection of DNA
sequences derived from genetically modified foodstuffs. They can be applicable also for PCR methods
used for detection of other target sequences in foodstuffs, e.g. for species detection and identification.
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.
EN ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions (ISO 24276)
ISO 16577, Molecular biomarker analysis — Terms and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 and EN ISO 24276 and
the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
probability of detection
POD
probability of a positive analytical outcome of a qualitative method for a given matrix at a given
concentration
Note 1 to entry: For a qualitative real-time PCR method it describes the probability that, for a given number of
DNA copies of the target sequence, PCR amplification will take place.
3.2
laboratory standard deviation
σ
L
expression of the standard deviation between laboratories which describes the dispersion of the log-
transformed laboratory-specific values for the LOD95%
3.3
mean amplification probability
λ
probability that, for a randomly selected DNA copy of the target sequence, PCR amplification will occur
3.4
slope parameter
b
slope of the POD curve (across laboratories) that indicates the deviation from the ideal POD curve (with
b = 1)
Note 1 to entry: The ideal POD curve is based on the assumption that the mean amplification probability is
independent of the number of DNA copies of the target sequence.
3.5
PCR efficiency
measured amplification rate for a DNA copy of the target sequence per PCR cycle in relation to the
theoretically achievable value of 1
Note 1 to entry: The PCR efficiency is calculated from the slope of a standard curve resulting from the decadic
semi-logarithmic plot of quantification cycle (Cq) values over the DNA concentration. The slope from the
calculated regression line can be used. The PCR efficiency can either be expressed as absolute number or as
percentage.
3.6
limit of detection
LOD
95%
mean number of copies of the target sequence yielding a probability of detection of 0,95
4 Principle
At the first step, a qualitative PCR method shall be single-laboratory validated and needs to show
satisfactory performance characteristics, see CEN/TS 17329-1.
As next step of the validation process, an inter-laboratory (or collaborative) validation study is
undertaken to assess the methods performance.
According to appropriate guidelines [1], [2], the main criterion in the validation of a qualitative real-
time PCR method by means of a collaborative study concerns the determination of the false-positive
rate and false-negative rate. Due to the use of different real-time PCR equipment from one laboratory to
the next, additional information on the robustness of the method can also be derived. Moreover, the
probability of detection (POD) of qualitative PCR methods can be evaluated, if the design of the
collaborative study is appropriate [3].
5 Validation of the performance characteristics by means of a collaborative
study
5.1 General
Guidance for conducting a collaborative validation study of qualitative PCR methods (i.e. organization,
protocol, number of participating laboratories etc.) and the description of all required components is
provided in other relevant documents [1]. Participants should have the required laboratory equipment
and proficiency in PCR testing.
The reagents essential for the PCR (oligonucleotides, PCR master mix) should be supplied to the
participants in order to ensure that different PCR reagents, which have not been checked for suitability,
do not influence the results.
Information about the results and data obtained in the study concerning the performance
characteristics shall be reported.
It is recommended that a small-scale collaborative study (pre-validation study involving 2 to 4
laboratories) is performed to test the general transferability of the method before the expenses of
organizing a large scale trial are incurred.
According to experiences and statistical considerations it is recommended that at least 12 laboratories
participate in the validation study.
5.2 False-positive rate and false-negative rate
5.2.1 General
Prepare a series of replicates of known negative test samples and of known positive test samples from
reference materials. If pure reference materials are not available, other sources as negative and positive
materials may be used.
Each participant receives the same number of encoded positive and negative samples. The positive test
samples contain defined quantities of the target DNA sequence of the positive material. The negative
samples only contain non-target DNA or matrix-specific negative material.
Each participant receives at least 6 positive and 6 negative samples, which have been encoded
beforehand. The participants perform the PCR measurements in single determination. Thus, for each
laboratory, at least 6 results for positive and 6 results for negative DNA samples are available for the
evaluation.
Requirements for preparation and evaluation of the homogeneity of replicate test samples are
described e.g. in [4].
5.2.2 Procedure with DNA as collaborative study test samples
In general, the test material used in collaborative studies of qualitative PCR methods consists of DNA
solutions.
The DNA is extracted from the sample material (in general from reference material) at a central facility
involved in the conduct of the study. This central laboratory also performs pre-tests with respect to the
quality of the extracted DNA (absence of PCR inhibition, amplificability, homogeneity). Guidance is
given in other relevant documents [2] [5].
The positive DNA samples should contain at least twice the copy number corresponding to the limit of
detection (LOD ) as determined in the course of the single-laboratory validation. A copy number of
95%
less than 20 copies of the
...
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