Foodstuffs - General guidelines for the validation of qualitative real-time PCR methods - Part 1: Single-laboratory validation

This document describes the performance characteristics and minimum performance criteria for conducting a single-laboratory validation study for qualitative (binary) real-time polymerase chain reaction (PCR) methods applied for the detection of specific DNA sequences present in foods.
The protocol was developed for qualitative real-time PCR methods for the detection of DNA sequences derived from genetically modified foodstuffs. It is applicable also for single-laboratory validation of qualitative PCR methods used for analysis of other food materials, e.g. for species detection and identification.
The document does not cover the evaluation of the applicability and the practicability with respect to the specific scope of the PCR method.

Lebensmittel - Allgemeine Anleitung für die Validierung qualitativer Realtime-PCR-Verfahren - Teil 1: Einzellaborvalidierung

Dieses Dokument legt die Leistungsmerkmale und Mindestleistungskriterien für die Durchführung einer Einzellabor-Validierungsstudie für Verfahren der qualitativen (binären) Realtime-Polymerasekettenreaktion (PCR) zum Nachweis spezifischer DNA-Sequenzen in Lebensmitteln fest.
Diese Vorschrift wurde entwickelt für qualitative Realtime-PCR-Verfahren zum Nachweis von DNA-Sequenzen, die von gentechnisch modifizierten Lebensmitteln stammen. Darüber hinaus ist es anwendbar für die Einzellaborvalidierung von qualitativen PCR-Verfahren, die zur Analyse sonstiger Lebensmittel eingesetzt werden, z. B. für den Nachweis und die Identifizierung von Spezies.
Das Dokument befasst sich nicht mit der Evaluierung der Anwendbarkeit und der Durchführbarkeit im Zusammenhang mit dem spezifischen Anwendungsbereich des PCR-Verfahrens.

Denrées alimentaires - Lignes directrices générales pour la validation des méthodes de PCR qualitative en temps réel - Partie 1 : Validation intralaboratoire

Le présent document décrit les caractéristiques de performance et les critères de performance minimaux pour la réalisation d’une étude de validation intralaboratoire relative aux méthodes de réaction en chaîne par polymérase (PCR) qualitative (binaire) en temps réel appliquées pour la détection de séquences d’ADN spécifiques présentes dans les aliments.
Le protocole a été mis au point pour les méthodes de PCR qualitative en temps réel appliquées pour la détection de séquences d’ADN extraites de produits alimentaires génétiquement modifiés. Il est également applicable à la validation intralaboratoire des méthodes de PCR qualitative utilisées pour l’analyse d’autres produits alimentaires, par exemple pour la détection et l’identification des espèces.
Le document ne couvre pas l’évaluation de l’applicabilité et de la faisabilité en ce qui concerne le domaine d’application spécifique de la méthode de PCR.

Živila - Splošne smernice za validacijo kvalitativnih metod PCR v realnem času - 1. del: Validacija v posameznem laboratoriju

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Current Stage
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Due Date
23-Jun-2021
Completion Date
23-Jun-2021

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SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17329-1:2021
01-marec-2021

Živila - Splošne smernice za validacijo kvalitativnih metod PCR v realnem času - 1.

del: Validacija v posameznem laboratoriju

Foodstuffs - General guidelines for the validation of qualitative real-time PCR methods -

Part 1: Single-laboratory validation

Lebensmittel - Allgemeine Anleitung für die Validierung qualitativer Realtime-PCR-

Verfahren - Teil 1: Einzellaborvalidierung

Denrées alimentaires - Lignes directrices générales pour la validation des méthodes de

PCR qualitative en temps réel - Partie 1 : Validation intralaboratoire
Ta slovenski standard je istoveten z: FprCEN/TS 17329-1
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
kSIST-TS FprCEN/TS 17329-1:2021 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17329-1:2021
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kSIST-TS FprCEN/TS 17329-1:2021
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17329-1
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
January 2021
ICS 67.050 Will supersede CEN/TS 17329-1:2019
English Version
Foodstuffs - General guidelines for the validation of
qualitative real-time PCR methods - Part 1: Single-
laboratory validation

Denrées alimentaires - Lignes directrices générales Lebensmittel - Allgemeine Anleitung für die

pour la validation des méthodes de PCR qualitative en Validierung qualitativer Realtime-PCR-Verfahren -

temps réel - Partie 1 : Validation intralaboratoire Teil 1: Einzellaborvalidierung

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, 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 NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17329-1:2021 E

worldwide for CEN national Members.
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FprCEN/TS 17329-1:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 6

5 Single-laboratory validation of the performance characteristics ................................................. 6

6 Validation report .......................................................................................................................................... 10

Annex A (informative) Estimation of the number of copies of the target sequence ......................... 11

Annex B (informative) Determination of limit of detection, precision and PCR efficiency ............ 13

B.1 General ............................................................................................................................................................. 13

B.2 Dilution series of the target DNA ............................................................................................................ 13

B.3 Preparation of PCR replicates ................................................................................................................. 13

B.4 Preparation of concentration levels for determination of the limit of detection

(LOD ) ........................................................................................................................................................ 13

95 %

B.5 Assessment of the observed variability of the measured number of copies around

the limit of detection (optional) ............................................................................................................. 14

B.6 Determination of the PCR efficiency (optional) ................................................................................ 15

B.6.1 General ............................................................................................................................................................. 15

B.6.2 Calculation of the PCR efficiency by means of linear regression analysis ............................... 15

Annex C (informative) Statistical Model ........................................................................................................... 17

C.1 POD and Poisson distribution ................................................................................................................. 17

C.2 POD, amplification probability and LOD ..................................................................................... 18

95 %

C.3 POD curve ........................................................................................................................................................ 18

C.4 Software tool and source code ................................................................................................................ 19

Annex D (informative) Robustness testing ...................................................................................................... 22

Bibliography ................................................................................................................................................................. 23

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European foreword

This document (FprCEN/TS 17329-1:2021) 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 document will supersede CEN/TS 17329-1:2019.

This new version was updated by inclusion of an option (in Annex C, C.4) to calculate the results of a

single-laboratory validation by using the R-package POD [15]. This calculation tool can make the user of

the Technical Specification independent from consultation of a statistician or a professional statistical

service provider when evaluating the results of a single-laboratory validation. The R-package can be

downloaded without being charged.
This series consists of two parts:
— Part 1: Single-laboratory validation;
— Part 2: Collaborative study.
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FprCEN/TS 17329-1:2021 (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 a newly developed food analytical method is fit-for-purpose and meets

certain performance characteristics and quality criteria as demonstrated by a particular set of validation

experiments.

The data determined by the single laboratory validation are the basis for the decision to apply a method

in-house. Furthermore, it helps to decide whether the method in question should be fully validated in the

framework of a collaborative study.

The aim of this document is to provide a protocol for single-laboratory validation 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 laboratories. 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 describes the performance characteristics and minimum performance criteria which

should be taken into account when conducting a single-laboratory validation study for qualitative

(binary) real-time polymerase chain reaction (PCR) methods applied for the detection of specific DNA

sequences present in foods.

The protocol was developed for qualitative real-time PCR methods for the detection of DNA sequences

derived from genetically modified foodstuffs. It is applicable also for single-laboratory validation of

qualitative PCR methods used for analysis of other food materials, e.g. for species detection and

identification.

The document does not cover the evaluation of the applicability and the practicability with respect to the

specific scope of the PCR method.
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 21571:2005, Foodstuffs - Methods of analysis for the detection of genetically modified organisms

and derived products - Nucleic acid extraction (ISO 21571:2005)

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 https://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.
As impacted by EN ISO 21571:2005/A1:2013.
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3.2
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.

3.3
limit of detection
LOD
95%

mean number of copies of the target sequence yielding a probability of detection of 0,95

4 Principle

Specific primers and also probes, depending on the detection system applied, have been designed for

specific amplification of a DNA target sequence by a qualitative real-time PCR method. As next step the

methods performance characteristics needs to be assessed to show that the method complies with the

quality criteria stipulated in relevant documents [1] [2].

According to the published guidelines the main criteria in the single-laboratory validation of a qualitative

real-time PCR method mainly concerns the limit of detection (at which the probability of detection

is ≥ 95 %), the specificity for the DNA target sequence and the robustness to small but deliberate

variations in the method parameters.

On the basis of the validation data, it can be verified whether the minimum required performance criteria

are fulfilled. This will be the basis for the applicability of the method by a single laboratory. A further

decision whether to conduct a validation of the method in the framework of a collaborative study can

then be taken.

Determination of the reproducibility (inter-laboratory transferability) and how the method performs at

different laboratories, in particular the false-positive/false-negative rate obtained with negative/positive

test samples, and the probability of detection (POD) across laboratories are evaluated by a collaborative

study, if the design is appropriate [3].

General guidelines for conducting a collaborative validation study are provided in Part 2 of this Technical

Specification.
5 Single-laboratory validation of the performance characteristics
5.1 General

Guidance for compiling the information required for complete and detailed description of all components

that should be provided with the protocol of the qualitative PCR methods (i.e. oligonucleotide sequences,

amplicon length, instrument or chemistry specifications, PCR conditions, analytical controls, etc.) is

described in other relevant documents [1] [2].

DNA extraction shall be according to the requirements and procedures specified in EN ISO 21571.

5.2 Limit of detection (LOD )
95 %

In qualitative PCR analysis (especially for the detection of genetically modified foodstuffs), the limit of

detection is usually defined as the amount of the target DNA at which an amplification product is detected

with a probability of at least 0,95 (LOD ). It is expressed in the number of copies of the target

95 %
sequence.
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The LOD should be determined by means of a dilution series of the target DNA, using a uniform

95 %
concentration of non-target DNA (background DNA) for each dilution level.

Annex A provides additional detailed information regarding the copy number estimation of the target

DNA. Annex C provides the basics of the specific statistical model adapted for PCR methods.

For each dilution level, perform 12 PCR replicate measurements. The lowest dilution level (i.e. the

lowest number of copies) for which all 12 replicates are positive is considered to be an approximate value

for LOD (see B.2). The LOD of the qualitative real-time PCR method should not exceed 20 copies

95 % 95 %
of the target sequence.

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 ; [4]) and the genome weight

[5] [6] [7]. 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 [8].

The quality and the concentration (very high or very low) of the background DNA used for the dilution

can influence the validation experiment. It is therefore highly recommended to use DNA tested for the

absence of PCR inhibitors (e.g. commercial molecular biology grade DNA preparations) and a

concentration which is relevant for DNA extracted from food.
Practical guidance which has been experimentally proven is given in Annex B.
5.3 Evaluation of data for the limit of detection (LOD )
95 %

Determine the LOD , the mean POD curve, and the 95 % confidence interval by means of a statistical

95 %

model, e.g. the complementary log-log model and the likelihood ratio test [3]. Details on the statistical

model are given in Annex C. For the calculation, the nominal copies added to the PCR reaction, the number

of replicates and the number of positive results are required.

The complementary log-log model corresponds to applications where we observe either zero events (e.g.

defects) or one or more events where the number of events is assumed to follow the Poisson distribution.

The LOD , the 95 % confidence interval and the mean POD curve along with the corresponding 95 %

95 %

confidence range can be calculated via a web service [9] or by using the R package POD [15].

Check the LOD for plausibility. A value significantly smaller than 2,996 suggests that the number of

95 %

copies of the target sequence that were actually added to the PCR reaction did not correspond to the

(nominal) numbers of copies estimated for the DNA solutions [3].

If more than two results are positive at the level with 0,1 copies of the target sequence per PCR, then the

DNA dilutions cannot be considered as verified and the number of copies has to be re-examined.

NOTE 1 The calculation of LOD is only valid if false-positive results are negligible, i.e. if the specificity testing

95 %
was successful and PCR carry-over contamination can be excluded.

NOTE 2 The level which will be the result of a tenfold dilution of 1 nominal copy is designated “level with 0,1

copies per PCR” for the sake of better readability throughout this document.
The statistical approach is described in [4].

This document is relevant for the validation of new methods. However, for method verification, 10 replicates

can be sufficient.
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5.4 PCR efficiency and variability of the measured copy number around the LOD
95 %

For the optional determination of copy numbers around the limit of detection, assign the copy numbers

to the respective Cq values on the basis of an additional calibration series of target DNA (preparation, see

Annex B, Table B.1).

In addition, the variability of the measured number of copies around LOD can be assessed (see B.5).

95 %

To this end, compare the repeatability standard deviations to the theoretical values resulting from the

Poisson model.

The experimental data also allow the calculation of the PCR efficiency (see B.6), the slope and the

coefficient of determination.
5.5 Specificity
5.5.1 General

Theoretical and experimental results from testing the method with the sequence databases and material

containing the target sequence should be provided. If available, this testing should include all relevant

and representative materials according to the scope of the method.
5.5.2 Theoretical test for specificity

Computer-aided (in-silico) specificity tests shall be carried out, examining the oligonucleotide and the

amplicon sequences with available bioinformatics tools (e.g. primer-dimer formation with primer3 [10]).

The homology to other sequences shall be tested by searches in nucleic acid sequence databases (e.g.

BLAST in GenBank [11]).

The in-silico analysis should not show any unwanted similarities between sequences which could

influence the analytical result. The oligonucleotide sequence(s) should be adapted accordingly, if

appropriate.
5.5.3 Practical test for specificity

Perform tests for unexpected cross-reactions with non-target DNA. Check the PCR detection system for

cross-reactivity with DNA from organisms that have similar (homologous) genetic elements, genes or

genetic constructs. Also check for species which are often present in food, e.g. as ingredient (corn, soya,

rape seed, rice, potato, wheat, cattle, chicken, pig, sheep, turkey, horse).

If non-target DNA is tested and a negative result is expected, at least 2500 copies should be added to the

PCR reaction, if possible. If no reference material with sufficiently high concentrations of the non-target

DNA is available, lower concentrations can be used and the number of copies added should be indicated.

Verify the amplifiability of the non-target DNA by means of an independent test.

Perform tests with target DNA. Add target DNA for which a positive result is expected in copy numbers

in the range of the LOQ (here: the copy number for LOD multiplied by a factor of 3, i.e. in general 20

95 %

to 60 copies per PCR). Add non-target DNA in a concentration of 100 ng/25 µl to 200 ng/25 µl of PCR mix

to the target DNA, in order to simulate conditions which are relevant in practice and could influence the

outcome.

It is sufficient to carry out each of the PCR tests for inclusivity (using target DNA) and exclusivity (using

non-target DNA) in duplicate determination.

In the experimental test, all the PCR results should fulfil the theoretical expectations.

If there is cross-reactivity which is considered to be acceptable, it should be indicated and taken into

account in the scope of the method.
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5.5.4 Robustness

In the single-laboratory validation, evaluate the robustness of a qualitative real-time PCR method

concerning different types of real-time PCR equipment, PCR reagent kits, annealing temperature applied

in the thermal cycling programme, the master mix volume and the primer and probe concentrations

(Table 1).

Implement a multifactorial experimental design [12]. The PCR reactions with the different combinations

of factors are done with target DNA at a concentration around the number of copies corresponding to the

LOD multiplied by a factor of 3 (corresponding to approximately 20 to 60 copies per PCR). Dilute the

95 %

target DNA in non-target DNA (background DNA, e.g. 20 ng/µl). For each factor-level combination, PCR

tests should at least be performed in triplicate. An example of the procedure is given in Annex D.

The method shall yield positive results for all combinations despite the modified conditions.

In the case of negative results, the PCR test for the corresponding combinations should be repeated. In

the case of repeated negative results, the method is not sufficiently robust and needs to be optimized.

Considerable deviations between Cq values could be an indication that the robustness of the method is

insufficient.

Table 1 — Robustness test of factors and modifications in the procedure conditions of

qualitative real-time PCR methods
Factor 1 0
PCR equipment A B
PCR master mix X Y
Primer concentration unchanged −30 %
Probe concentration unchanged −30 %
Volume of PCR reagent 19 µl of PCR reagent mix 21 µl of PCR reagent mix
mix
+ 5 µl of DNA + 5 µl of DNA
(if total volume is 25 µl)
Annealing temperature +1 °C −1 °C
Table 2 — Robustness test by means of an orthogonal experimental design
Factor Combination
1 2 3 4 5 6 7 8
PCR equipment 1 1 1 1 0 0 0 0
PCR master mix 1 1 0 0 1 1 0 0
Primer concentration 1 0 1 0 1 0 1 0
Probe concentration 1 0 0 1 0 1 1 0
Total volume of PCR reagent 1 1 0 0 0 0 1 1
mix
Annealing temperature 1 0 1 0 0 1 0 1
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The orthogonal experimental design is characterized by the fact that, for any pair of factors (e.g. PCR

kit/primer concentration), all four factor-level combinations (1+1, 1+0, 0+1, 0+0) occur with the same

frequency (twice each) (Table 2). This setup guarantees that possible factorial interaction effects on the

PCR test results can also be detected.
6 Validation report

Information about the results and data obtained by the single-laboratory validation of the performance

characteristics shall be compiled in a report.
This report should comprise as minimum:

— Concerning the LOD : the species of the target DNA; information on how the copy number was

95 %

assessed for the sensitivity tests; estimated LOD ; type and quantity of background DNA used

95 %

— Concerning the theoretical specificity tests: results, database used and date when the database was

assessed

— Concerning practical specificity tests: species from which the DNA was extracted and quantity used

per reaction, results, type and quantity of background DNA

— Concerning robustness: species from which the DNA was extracted and quantity per reaction,

parameters changes, results.
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Annex A
(informative)
Estimation of the number of copies of the target sequence

The theoretical 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 ) and the genome

weight available in the literature or in different databases [5] [6] [7]. The reference values and the

reference used shall be indicated. A list of genome weights of the most relevant species is provided.

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 [8].
Table A.1 — Mass of the haploid genome of relevant species
Species Mass of haploid
genome
(in pg)
Cotton 2,33 [5]
Barley 5,55 [6]
Potato 1,8 [5]
Salmon 3,27 [7]
Linseed 0,70 [6]
Alfalfa 1,57 [5]
Corn 2,6 [5]
Papaya 0,39 [5]
Rapeseed (Brassica napus) 1,15 [6]
Rice 0,45 [6]
Soya 1,13 [6]
Common wheat 17,33 [6]
Sugar beet 1,25 [6]
Cattle 3,57 [7]
Chicken 1,25 [7]
Pig 2,98 [7]
Sheep 3,33 [7]
Turkey 1,40 [7]
Horse 3,38 [7]
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When using genomic DNA, first of all calculate the number of genome equivalents, G per microlitre on

the basis of the mass of the respective haploid genome of the species according to Formula (A.1):

C × 1000
G = (A.1)
where
C is the DNA concentration, in ng/µl;
H is the mass of the haploid genome, in pg.

The corresponding number of copies of the target sequence can be estimated on the basis of the number

of genome equivalents. In this context, the number of copies per haploid genome as well as possible

differences in the ploidy level and the zygosity of the employed reference material have to be taken into

account.

When using plasmid DNA or amplicon DNA, the number of copies N can be calculated on the basis of the

length of the sequence (in base pairs, bp) and the DNA concentration determined spectrophotometrically

or fluorometrically (in ng/μl) according to Formula (A.2):
N ××6022 10 (A.2)
l × 660
where
is the DNA mass concentration, in ng/µl;
l is the length of the sequence, in base pair (bp).

Plasmid DNA should be linearized by means of restriction digest before being used in the PCR.

Reference DNA or reference material with a certified percentage, 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.
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Annex B
(informative)
Determination of limit of detection, precision and PCR efficiency
B.1 General

In the following, an exemplary work schedule for the experimental determination of LOD (in copies

95 %

of the target sequence), of precision data and of the PCR efficiency is described.

B.2 Dilution series of the target DNA

Prepare a dilution series of the target DNA, with a uniform concentration of non-target DNA for each

dilution level. Supplement the DNA dilutions of the target DNA (genomic DNA, plasmid DNA or amplicon

DNA) with sufficient background DNA (e.g. plant species DNA or salmon sperm DNA in concentrations of

up to 200 ng per 25 µl of PCR mix), thus stabilizing them for the PCR.

Before use, the background DNA shall be tested for inhibition with regard to the target sequence (e.g.

according to [14]).

To determine the LOD , PCR tests should be performed for the six concentration levels given as

95 %
examples in Table B.1, with 12 replicates each.

By including an additional calibration series, it is possible to obtain data regarding both the variability of

the measured number of copies around LOD and the PCR efficiency. For this purpose, prepare a

95 %

dilution series with at least 4 levels (with background DNA, if possible) from a standard DNA solution

(target DNA consisting of genomic DNA, plasmid DNA or amplicon DNA) (e.g. 2500, 500, 100, 50 copies

per reaction). Perform PCR tests for the different levels with at least 3 repli
...

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