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

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Published
Publication Date
09-Apr-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
10-Apr-2019
Completion Date
10-Apr-2019

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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.

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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.
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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

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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.
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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.
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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

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

probability that, for a randomly selected DNA copy of the target sequence, PCR amplification will occur

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3.4
slope parameter

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.
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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

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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.
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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

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).
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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

individual laboratories differ from one another. A value of σ = 1 should not be exceeded.

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.
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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
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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.
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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
...

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