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

Status
Published
Public Enquiry End Date
19-Jan-2019
Publication Date
26-May-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
18-Apr-2019
Due Date
23-Jun-2019
Completion Date
27-May-2019

Buy Standard

Technical specification
TS CEN/TS 17329-2:2019
English language
23 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day
Draft
kTS FprCEN/TS 17329-2:2019
English language
23 pages
sale 10% off
Preview
sale 10% off
Preview
e-Library read for
1 day

Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TS CEN/TS 17329-2:2019
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
SIST-TS CEN/TS 17329-2:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------

SIST-TS CEN/TS 17329-2:2019

---------------------- Page: 2 ----------------------

SIST-TS CEN/TS 17329-2:2019


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.

---------------------- Page: 3 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
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

2

---------------------- Page: 4 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
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.
3

---------------------- Page: 5 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
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.
4

---------------------- Page: 6 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
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
5

---------------------- Page: 7 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
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.
6

---------------------- Page: 8 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
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 target sequence per PCR reaction should not be used as positive test sample.
The test samples are to be prepared with up to 100 ng per 25 μl PCR reaction of suitable background
DNA. In case of inclusion of DNA extraction it will be a combined collaborative trial of DNA extraction
method and a real-time PCR method.
Negative DNA samples should contain only background DNA at the given concentration.
5.2.3 Procedure with food material as collaborative study test samples
If the target DNA sequence is to be detected mainly for one specific food matrix and if, for this matrix, no
validated extraction procedure is available, the study uses known positive and known negative sample
materials from which DNA shall be extracted by the participants.
For a pre-test, a central laboratory should extract DNA from the sample material using a defined DNA
extraction procedure and should pre-test the quality of the extracted DNA concerning absence of PCR
inhibition, amplificability and homogeneity [2] [5].
The participants shall check the performance of the DNA extraction method. For this purpose, an
additional sample for positive extraction control (P) is provided. This sample allows each participant to
test the extraction method and its own reagents. To this end, a PCR analysis is performed both for the
DNA from P and for a positive control DNA provided by the organizer. The analysis is carried out in
7

---------------------- Page: 9 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
duplicate and the mean Cq values are then compared. The mean Cq value for the DNA from P should not
exceed the mean Cq value for the positive control by more than 1 (for example: Cq value of positive
control is 23, then the Cq value of DNA from P should not exceed the value of 24). In case of GMO testing
methods this applies to the target-sequence PCR as well as to the taxon-specific PCR. Moreover, it is
recommended to carry out an inhibition control test [2] [5].
For screening PCR methods, samples from more than one relevant species (e.g. corn and soya) may be
included in the pool of test samples. The reagents for the PCR (oligonucleotides, PCR master mix etc.)
are provided by the organizer of the collaborative study.
5.2.4 Evaluation and performance criteria
On the basis of the available results, the false-positive rates, fp in %, and false-negative rates, fn in %,
are calculated with Formula (1) and (2):
mkn
fp 100 × (1)
tkn
where
mkn is the number of misclassified known negative samples;
tkn is the total number of known negative samples.
mkp
(2)
fn 100 ×
tkp
where
mkp is the number of misclassified known positive samples;
tkp is the total number of known positive samples.
The false-positive rate and false-negative rate assessed by the study should demonstrate that neither
the false-negative rate nor the false-positive rate exceeds 5 %.
If a false-positive rate above 5 % was observed it should be investigated on a case-by-case basis. The
method documentation should then provide relevant instructions. Apparently false positive results
could occur, for example, in qualitative PCR screening tests for the detection of genetic elements which
have a high inter-species sequence homology or occur naturally.
NOTE Annex A provides additional detailed information regarding the conduct of the collaborative study, the
preparation of test samples and the evaluation of the results and pertaining documentation.
5.3 Robustness
In the collaborative study, the robustness of a qualitative real-time PCR method is evaluated concerning
the different types of real-time PCR equipment that are used and the different laboratory conditions.
The method shall produce the expected results despite these changes. There shall not be any noticeable
difference between the results obtained using different real-time PCR equipment.
Robustness is primarily tested in the framework of single-laboratory validation. Additional parameters
including any pre-analytical influence by transport and time lag before starting the PCR tests are
assessed in frame of the collaborative study.
8
=
=

---------------------- Page: 10 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
5.4 Probability of detection (POD)
5.4.1 General
For the validation of the probability of detection (POD) of a qualitative real-time PCR method the
different performance characteristics (laboratory standard deviation σ , mean amplification
L
probability λ, slope parameter b, LOD ) are calculated on basis of data from a sufficient number of
95%
laboratories that assessed several replicates across a number of coy number concentrations (in the
range of the LOD ).
95%
5.4.2 Preparation of concentration levels
The participating laboratories receive a standard DNA with a calculated number of copies of the target
sequence. On the basis of this standard DNA, a dilution series with different concentration levels for the
target sequence is prepared.
Four concentration levels are chosen covering the dynamic range to obtain data for a standard curve.
For the validation of the POD parameters the copy number concentrations should be chosen carefully at
levels where the PCR probability of detection is < 1,0, e.g. at copy numbers equivalent to POD values of
0,05, 0,25, 0,5, 0,75, 0,95 and > 0,95. An example is given in Annex A, Table A.1.
Alternative procedures for preparation and providing the sample DNAs with different copy number
concentrations can be applied.
The number of copies of the target sequence can be calculated on basis of haploid genome equivalents
using the measured DNA concentration (see EN ISO 21571:2005, Annex B [6]) and the genome weight
[7] [8] [9]. The use of digital PCR equipment (e.g. digital droplet PCR) is an alternative approach which
allows an accurate determination of the number of copies of a target sequence or the concentration of a
DNA solution [10].
The dilutions are prepared in a buffer solution having a uniform non-target DNA concentration. For this
purpose, the standard DNA is added to the corresponding amount of background DNA and thereby
stabilized for the PCR.
5.4.3 Number of PCR replicates per laboratory
The specified replicate numbers for the given concentration levels represent the minimum number
necessary to obtain a sufficient statistical reliability for the LOD and for the corresponding
95%
precision data [3].
Each laboratory performs PCR measurements in triplicate determination for the standard curve levels.
For the POD curve six replicate determinations are performed with each copy number level.
5.4.4 Evaluation of POD data
On the basis of the standard curve, the values for the slope and the coefficient of determination of the
calibration function are calculated for each laboratory and are presented in a table (see Annex A, Table
A.3).
A calibration function slope of approximately -3,1 to -3,6 is considered to be an indication of good PCR
efficiency. The coefficient of determination should be at least 0,98.
The numbers of positive qualitative PCR results obtained for the six concentration levels in the low copy
number range are tabulated (Annex A, Table A.2).
On the basis of the qualitative data, the laboratory-specific POD curves can be calculated (Annex A,
Figure A.1).
9

---------------------- Page: 11 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
Plausibility checks and outlier tests (e.g. according to Grubbs) are carried out for the laboratory-specific
amplification probabilities λ and slope parameters b (B.4).
The value calculated for b should be in the range 0,65 < b ≤ 2 in order to ensure sensitive POD curves.
This range is based on the condition that the LOD value is not >20 copies and that the amplification
95%
probability in the range from 0,1 to 100 copies is not >1.
The mean amplification probability λ and the corresponding 95 % confidence interval are calculated
and tabulated with the slope parameter b of the POD curve across laboratories, laboratory standard
deviation (σ ) as well as the LOD (in copies of the target sequence for the theoretical median
L 95%
laboratory at POD = 0,95). If no statistically significant deviation for b = 1 is observed, the calculation of
the other performance characteristics can be done using this value [3].
The calculated values for the POD curve across laboratories along with the corresponding prediction
intervals as well as the rates of detection for the different concentration levels of the target sequence
can be also presented graphically (Annex A, Figure A.2).
Once the lab-specific POD curves have been calculated, the values for the other performance
characteristics can be calculated and tabulated (Annex A, Table A.5; Annex B).
NOTE 1 A value for b <1 indicates that the PCR amplification is inhibited already for low numbers of copies.
NOTE 2 A value of λ > 1 is an indication that the true number of copies of the standard DNA is actually higher
and the nominal value used for the calculation was not correct (e.g. by incorrect DNA concentration measurement
beforehand).
5.4.5 Performance criteria for the POD curve
The upper 95 % prediction limit of the laboratory-specific LOD values should not exceed 20 copies
95%
of the target sequence. Otherwise the sensitivity (and corresponding PCR efficiency) of the PCR
methods is not satisfactory.
As a general rule, the LOD is subject to considerable variability across laboratories. For this reason,
95%
a test is carried out on the basis of the ratio of the 5 % and 95 % quantiles. This ratio should not exceed
a value of 5.
The laboratory standard deviation σ describes, in the log domain, how much the POD curves of the
L
individual laboratories differ from one another. A value of σ = 1 should not be exceeded.
L
NOTE 1 If LOD is less than 4 copies for 5 % of all laboratories, lies in the range of 4 to 20 copies for 90 % of
95%
all laboratories and exceeds 20 copies for the remaining 5 % of all laboratories, this ratio has a value of 5.
NOTE 2 With a value of 5 for the ratio of the upper and lower prediction limits of the laboratory-specific
LOD values, the mean amplification probability λ is approximately 15 % (an estimate based on first
95%
calculating the mean LOD value corresponding to a distribution of LOD values with 3 copies as 5 %
95% 95%
quantile and 15 copies as 95 % quantile, and then on the relationship derived from the Poisson distribution and
λ × LOD = 2,996).
95%
NOTE 3 In the case of a value for σ exceeding 1, the LOD (corresponding to a ‘theoretical’ median
L 95%
laboratory) exceeds the limit of 20 copies, and the ratio of the upper and lower prediction limits of the laboratory-
specific LOD values exceeds the value of 50 copies. In other words, a value of σ >1 means that the method
95% L
has a very poor mean sensitivity and, moreover, that sensitivity can be expected to be subject to considerable
variability from one laboratory to the other.
10

---------------------- Page: 12 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
6 Calculation of precision data for test samples
The standard curves make it possible to calculate copy numbers for the test samples. Moreover,
precision data for the statistical parameters (mean, repeatability and reproducibility standard
deviation, etc.) according to ISO 5725-2 can be calculated (Annex A, Table A.6).
In connection with the calculation of these precision data, it is necessary to identify and eliminate
possible outlier data sets.
7 Study report
Information about the results and data obtained by the collaborative study should be compiled in a
report.
This report should comprise as minimum:
— Number of participating laboratories; number of laboratories which received samples; number of
laboratories which returned results; number of laboratories excluded from the evaluation
(Annex A, Table A.4)
— Description of materials provided to the participants
— Description of the sample material (source, type and quality of positive samples; information on
how the copy number was assessed; concentration of the target DNA; type and quality of
background DNA)
— Description of source, type and quality of material used for preparation of concentration levels
— Number of all results, of accepted results, of outliers
— Results concerning false-positive and false-negative rate, sensitivity
11

---------------------- Page: 13 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
Annex A
(informative)

Instructions for the conduct of the collaborative study
A.1 Collaborative study samples and standard DNA
A.1.1 DNA solutions containing the target sequence
Reference DNA or reference material with a certified quantification of the target DNA, which is
characterized as well as possible with regard to the target sequence, e.g. regarding the number of copies
and zygosity, is especially well suited.
A.1.2 DNA solutions not containing the target sequence
Reference DNA or reference material which does not contain the respective target DNA and which, for
example in case of GMO, corresponds to the isogenic lines, can be used.
It shall be be kept in mind that the certification of a reference material only refers to the indicated
material content. This means that contaminations with other materials (e.g. GMOs) at trace level (<
0,1 %) are still possible. For this reason it can be an advantage for the production of positive or negative
DNA samples to use e.g. material from individual organisms, which have been proven not to contain the
target sequence.
In case of GMO testing methods, usually for each species analyzed in the collaborative study, one non-
genetically modified material is analyzed.
If background DNA is used, it has to be checked for PCR inhibition before use.
A.1.3 Standard DNA
Standard DNA can be produced from genomic DNA, plasmid DNA or amplicon DNA. Sufficient standard
DNA shall be provided to allow for the preparation of the dilution series with at least 4 concentration
levels presented in Table A.1. Ideally, enough volume is produced to enable each participating
laboratory to repeat the preparation of the dilution series if necessary.
For the preparation of the dilution series, a buffer solution with background DNA (at least 20 ng/μl of a
non-target DNA, e.g. salmon sperm DNA) is to be provided.
12

---------------------- Page: 14 ----------------------

SIST-TS CEN/TS 17329-2:2019
CEN/TS 17329-2:2019 (E)
Table A.1 — Example of a scheme for the preparation of a dilution series
Dilution Preparation (example) Number of copies of Number of
level target sequence PCR replicates
(in 5 µl)
1 a 2 500 3
dilute DNA stock solution with 0,2 x TE
2 a 500 3
10 µl (500 copies/µl) + 40 µl 0,2 x TE
3 a 100 3
20 µl (100 copies/µl) + 80 µl 0,2 x TE
4
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17329-2:2019
01-januar-2019
äLYLOD6SORãQHVPHUQLFH]DYDOLGDFLMRNYDOLWDWLYQLKPHWRG3&5YUHDOQHPþDVX
GHO0HGODERUDWRULMVNDãWXGLMD
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
Ta slovenski standard je istoveten z: FprCEN/TS 17329-2
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
kSIST-TS FprCEN/TS 17329-2:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019

---------------------- Page: 2 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019


FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17329-2
SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION

November 2018
ICS 67.050
English Version

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


This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 275.

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.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.


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. FprCEN/TS 17329-2:2018 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
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 . 10
7 Study report . 10
Annex A (informative) Instructions for the conduct of the collaborative study . 12
A.1 Collaborative study samples and standard DNA . 12
A.1.1 DNA solutions containing the target sequence . 12
A.1.2 DNA solutions not containing the target sequence . 12
A.1.3 Standard DNA . 12
A.2 Reagents . 13
A.3 Documentation and evaluation . 13
A.3.1 General . 13
A.3.2 Results . 14
A.3.2.1 Calculations for each laboratory (in columns): . 14
A.3.2.2 Summary of results of all laboratories . 16
A.3.2.3 Six-fold blinded (encoded) samples . 18
A.3.2.4 Dilution series . 19
Annex B (informative) Statistical model . 20
B.1 POD and Poisson distribution . 20
B.2 POD, amplification probability and LOD . 21
95%
B.3 POD curve . 21
B.4 Statistical calculation of the POD model . 21
Bibliography . 23

2

---------------------- Page: 4 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
European foreword
This document (FprCEN/TS 17329-2:2018) has been prepared by Technical Committee CEN/TC 275
“Food analysis - Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.
This Technical Specification consists of two parts,
— Part 1: Single-laboratory validation
— Part 2: Collaborative study
3

---------------------- Page: 5 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
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.
4

---------------------- Page: 6 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
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)
5

---------------------- Page: 7 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
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 and 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 (wi 335)xxxx-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 by the study concerning the performance criteria 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 expense of
organizing a large scale trial is incurred.
According to experiences and statistical considerations it is recommended that at least 12 laboratories
participate in the validation study.
6

---------------------- Page: 8 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
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 target sequence per PCR reaction should not be used as positive test sample.
The test samples are to be prepared with up to 100 ng per 25 μl PCR reaction of suitable background
DNA. In case of inclusion of DNA extraction it will be a combination collaborative trial of DNA extraction
method and a real-time PCR method.
Negative DNA samples should contain only background DNA at the given concentration.
5.2.3 Procedure with food material as collaborative study test samples
If the target DNA sequence is to be detected mainly for one specific food matrix and if, for this matrix, no
validated extraction procedure is available, the study uses known positive and known negative sample
materials from which DNA shall be extracted by the participants.
For a pre-test, a central laboratory should extract DNA from the sample material using a defined DNA
extraction procedure and should pre-test the quality of the extracted DNA concerning absence of PCR
inhibition, amplificability and homogeneity [2] [5].
The participants shall check the performance of the DNA extraction method. For this purpose, an
additional sample for positive extraction control (P) is provided. This sample allows each participant to
test the extraction method and its own reagents. To this end, a PCR analysis is performed both for the
DNA from P and for a positive control DNA provided by the organizer. The analysis is carried out in
duplicate and the mean Cq values are then compared. The mean Cq value for the DNA from P should not
exceed the mean Cq value for the positive control by more than 1 (for example: Cq value of positive
control is 23, then the Cq value of DNA from P should not exceed the value of 24). In case of GMO testing
methods this applies to the target-sequence PCR as well as to the taxon-specific PCR. Moreover, it is
recommended to carry out an inhibition control test [2] [5].
7

---------------------- Page: 9 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
For screening PCR methods, samples from more than one relevant species (e.g. corn and soya) may be
included in the pool of test samples. The reagents for the PCR (oligonucleotides, PCR master mix etc.)
are provided by the organizer of the collaborative study.
5.2.4 Evaluation and performance criteria
On the basis of the available results, the false-positive rates, fp in %, and false-negative rates, fn in %,
are calculated with Formula (1) and (2):
mkn
fp 100 × (1)
tkn
where
mkn is the number of misclassified known negative samples;
tkn is the total number of known negative samples.
mkp
fn 100 × (2)
tkp
where
mkp is the number of misclassified known positive samples;
tkp is the total number of known positive samples.
The false-positive rate and false-negative rate assessed by the study should demonstrate that neither
the false-negative rate nor the false-positive rate exceeds 5 %.
If a false-positive rate above 5 % was observed it should be investigated on a case-by-case basis. The
method documentation should then provide relevant instructions. Apparently false positive results
could occur, for example, in qualitative PCR screening tests for the detection of genetic elements which
have a high inter-species sequence homology or occur naturally.
NOTE Annex A provides additional detailed information regarding the conduct of the collaborative study, the
preparation of test samples and the evaluation of the results and pertaining documentation.
5.3 Robustness
In the collaborative study, the robustness of a qualitative real-time PCR method is evaluated concerning
the different types of real-time PCR equipment that are used and the different laboratory conditions.
The method shall produce the expected results despite these changes. There shall not be any noticeable
difference between the results obtained using different real-time PCR equipment.
Robustness is primarily tested in the framework of single-laboratory validation. Additional parameters
including any pre-analytical influence by transport and time lag before starting the PCR tests are
assessed in frame of the collaborative study.
5.4 Probability of detection (POD)
5.4.1 General
For the validation of the probability of detection (POD) of a qualitative real-time PCR method the
different performance characteristics (laboratory standard deviation σ , mean amplification
L
probability λ, slope parameter b, LOD ) are calculated on basis of data from a sufficient number of
95%
laboratories that assessed several replicates across a number of coy number concentrations (in the
range of the LOD ).
95%
8
=
=

---------------------- Page: 10 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
5.4.2 Preparation of concentration levels
The participating laboratories receive a standard DNA with a calculated number of copies of the target
sequence. On the basis of this standard DNA, a dilution series with different concentration levels for the
target sequence is prepared.
Four concentration levels are chosen covering the dynamic range to obtain data for a standard curve.
For the validation of the POD parameters the copy number concentrations should be chosen carefully at
levels where the PCR probability of detection is < 1,0, e.g. at copy numbers equivalent to POD values of
0,05, 0,25, 0,5, 0,75, 0,95 and > 0,95. An example is given in Annex A, Table A.1.
Alternative procedures for preparation and providing the sample DNAs with different copy number
concentrations can be applied.
The number of copies of the target sequence can be calculated on basis of haploid genome equivalents
using the measured DNA concentration (see EN ISO 21571:2005, Annex B [6]) and the genome weight
[7] [8] [9]. The use of digital PCR equipment (e.g. digital droplet PCR) is an alternative approach which
allows an accurate determination of the number of copies of a target sequence or the concentration of a
DNA solution [10].
The dilutions are prepared in a buffer solution having a uniform non-target DNA concentration. For this
purpose, the standard DNA is added to the corresponding amount of background DNA and thereby
stabilized for the PCR.
5.4.3 Number of PCR replicates per laboratory
The specified replicate numbers for the given concentration levels represent the minimum number
necessary to obtain a sufficient statistical reliability for the LOD and for the corresponding
95%
precision data [3].
Each laboratory performs PCR measurements in triplicate determination for the standard curve levels.
For the POD curve six replicate determinations are performed with each copy number level.
5.4.4 Evaluation of POD data
On the basis of the standard curve, the values for the slope and the coefficient of determination of the
calibration function are calculated for each laboratory and are presented in a table (see Annex A, Table
A.3).
A calibration function slope of approximately -3,1 to -3,6 is considered to be an indication of good PCR
efficiency. The coefficient of determination should be at least 0,98.
The numbers of positive qualitative PCR results obtained for the six concentration levels in the low copy
number range are tabulated (Annex A, Table A.2).
On the basis of the qualitative data, the laboratory-specific POD curves can be calculated (Annex A,
Figure A.1).
Plausibility checks and outlier tests (e.g. according to Grubbs) are carried out for the laboratory-specific
amplification probabilities λ and slope parameters b (B.4).
The value calculated for b should be in the range 0,65 < b ≤ 2 in order to ensure sensitive POD curves.
This range is based on the condition that the LOD value is not >20 copies and that the amplification
95%
probability in the range from 0,1 to 100 copies is not >1.
The mean amplification probability λ and the corresponding 95 % confidence interval are calculated
and tabulated with the slope parameter b of the POD curve across laboratories, laboratory standard
deviation (σ ) as well as the LOD (in copies of the target sequence for the theoretical median
L 95%
laboratory at POD = 0,95). If no statistically significant deviation for b = 1 is observed, the calculation of
the other performance characteristics can be done using this value [3].
9

---------------------- Page: 11 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
The calculated values for the POD curve across laboratories along with the corresponding prediction
intervals as well as the rates of detection for the different concentration levels of the target sequence
can be also presented graphically (Annex A, Figure A.2).
Once the lab-specific POD curves have been calculated, the values for the other performance
characteristics can be calculated and tabulated (Annex A, Table A.5; Annex B).
NOTE 1 A value for b <1 indicates that the PCR amplification is inhibited already for low numbers of copies.
NOTE 2 A value of λ > 1 is an indication that the true number of copies of the standard DNA is actually higher
and the nominal value used for the calculation was not correct (e.g. by incorrect DNA concentration measurement
beforehand).
5.4.5 Performance criteria for the POD curve
The upper 95 % prediction limit of the laboratory-specific LOD values should not exceed 20 copies
95%
of the target sequence. Otherwise the sensitivity (and corresponding PCR efficiency) of the PCR
methods is not satisfactory.
As a general rule, the LOD is subject to considerable variability across laboratories. For this reason,
95%
a test is carried out on the basis of the ratio of the 5 % and 95 % quantiles. This ratio should not exceed
a value of 5.
The laboratory standard deviation σ describes, in the log domain, how much the POD curves of the
L
individual laboratories differ from one another. A value of σ = 1 should not be exceeded.
L
NOTE 1 If LOD is less than 4 copies for 5 % of all laboratories, lies in the range of 4 to 20 copies for 90 % of
95%
all laboratories and exceeds 20 copies for the remaining 5 % of all laboratories, this ratio has a value of 5.
NOTE 2 With a value of 5 for the ratio of the upper and lower prediction limits of the laboratory-specific
LOD values, the mean amplification probability λ is approximately 15 % (an estimate based on first
95%
calculating the mean LOD value corresponding to a distribution of LOD values with 3 copies as 5 %
95% 95%
quantile and 15 copies as 95 % quantile, and then on the relationship derived from the Poisson distribution and
λ × LOD = 2,996).
95%
NOTE 3 In the case of a value for σ exceeding 1, the LOD (corresponding to a ‘theoretical’ median
L 95%
laboratory) exceeds the limit of 20 copies, and the ratio of the upper and lower prediction limits of the laboratory-
specific LOD values exceeds the value of 50 copies. In other words, a value of σ >1 means that the method
95% L
has a very poor mean sensitivity and, moreover, that sensitivity can be expected to be subject to considerable
variability from one laboratory to the other.
6 Calculation of precision data for test samples
The standard curves make it possible to calculate copy numbers for the test samples. Moreover,
precision data for the statistical parameters (mean, repeatability and reproducibility standard
deviation, etc.) according to ISO 5725-2 can be calculated (Annex A, Table A.6).
In connection with the calculation of these precision data, it is necessary to identify and eliminate
possible outlier data sets.
7 Study report
Information about the results and data obtained by the collaborative study should be compiled in a
report.
This report should comprise as minimum:
10

---------------------- Page: 12 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
— Number of participating laboratories; number of laboratories which received samples; number of
laboratories which returned results; number of laboratories excluded from the evaluation
(Annex A, Table A.4)
— Description of materials provided to the participants
— Description of the sample material (source, type and quality of positive samples; information on
how the copy number was assessed; concentration of the target DNA; type and quality of
background DNA)
— Description of source, type and quality of material used for preparation of concentration levels
— Number of all results, of accepted results, of outliers
— Results concerning false-positive and false-negative rate, sensitivity
11

---------------------- Page: 13 ----------------------
kSIST-TS FprCEN/TS 17329-2:2019
FprCEN/TS 17329-2:2018 (E)
Annex A
(informative)

Instructions for the conduct of the collaborative study
A.1 Collaborative study samples and standard DNA
A.1.1 DNA solutions containing the target sequence
Reference DNA or reference material with a certified quantification of the target DNA, which is
characterized as well as possible with regard to the target sequence, e.g. regarding the number of copies
and zygosity, is especially well suited.
A.1.2 DNA solutions not containing the target sequence
Reference DNA or reference material which does not contain the respective target DNA and which, for
example in case of GMO, corresponds to the isogenic lines, can be used.
It shall be be kept in mind that the certification of a reference material only refers to the indicated
material content. This means that contaminations with other materials (e.g. GMOs) at trace level (<
0,1 %) are still possible. For this reason it can be an advantage for the production of positive or negative
DNA samples to use e.g. ma
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.