prEN 18000-3
(Main)Animal health diagnostic analyses - Control of in vitro diagnostic reagents - Part 3: Reagents for PCR techniques
Animal health diagnostic analyses - Control of in vitro diagnostic reagents - Part 3: Reagents for PCR techniques
This document specifies the control and approval of in vitro diagnostic reagents used in animal health for the detection, and/or absolute quantification of pathogen-specific nucleic acid (DNA or RNA) by PCR (e.g. endpoint PCR, real-time PCR, reverse transcription-PCR).
This document is applicable to diagnostic reagents as a priority for infectious diseases (due to bacteria, viruses, fungi, or parasites, including genetic markers associated with pathogenicity, such as antimicrobial resistance or toxin production) and associated animal species for which harmonization of practices in this area is needed, i.e. those for which the national, regional or international regulatory framework provides for the control of trade in animals and/or animal products and/or the definition of a health status (absence of infection) of areas, establishments or individuals. Anyhow, all reagents designated by the competent authorities fall under the scope of this document. Nevertheless, the authorities or any other animal health stakeholder can choose to derogate in specific and very limited situations such as emerging, exotic or rare diseases.
This document is not applicable to all existing diagnostic reagents, in particular those for which certain parameters described in this document cannot be validly evaluated in accordance with international requirements, due, e.g. to the absence of a specific reference standard and/or accessible and duly validated reference materials.
The PCR diagnosis usually involves the use of a nucleic acid extraction and/or purification reagent, and a PCR reagent. The PCR method (when applicable) involves the successive use of these distinct reagents. PCR reagent control can be performed if the applicant provides evidence of the validity of the PCR reagent for use in the animal health diagnostic analysis, by proving its diagnostic performances with nucleic acid extracts obtained from the different matrices described in the instruction for use. The control of a complete PCR method by the applicant and the control organization is performed only if the PCR reagent cannot be dissociated from an nucleic acid extraction and/or purification systems. This document does not cover the control of the nucleic acid extraction and/or purification reagents, only.
This document does not cover the step in which the user verifies a reagent (analysis method adoption).
NOTE Prion diseases are not included in the scope of this third part of the EN 18000 series. Unlike other infectious diseases, prion diseases are not diagnosed using PCR assays because prions lack a nucleic acid component and consist solely of an abnormally folded conformer of the normal host protein.
Tiergesundheitsdiagnostische Analysen - Kontrolle von in-vitro-diagnostischen Reagenzien - Teil 3: Reagenzien für PCR Verfahren
Dieses Dokument legt die Prüfung und Zulassung von im Bereich der Tiergesundheit verwendeten in-vitro-diagnostischen Reagenzien zum Nachweis und/oder zur absoluten Quantifizierung von erregerspezifischer Nukleinsäure (DNA oder RNA) durch PCR (z. B. Endpunkt-PCR, Real-Time-PCR, Reverse-Transkription-PCR) fest.
Dieses Dokument gilt für diagnostische Reagenzien, vorrangig für Infektionskrankheiten (durch Bakterien, Viren, Pilze oder Parasiten, einschließlich mit Pathogenität verbundener genetischer Marker, beispielsweise für antimikrobielle Resistenz oder Toxinproduktion) und entsprechende Tierarten, für die eine Harmonisierung der Praktiken in diesem Bereich erforderlich ist, d. h. für diejenigen, für die der nationale, regionale oder internationale Regelungsrahmen die Kontrolle des Handels mit Tieren und/oder tierischen Erzeugnissen und/oder die Festlegung eines Gesundheitsstatus (Infektionsfreiheit) von Gebieten, Einrichtungen oder Personen vorsieht. Gleichwohl fallen alle von den zuständigen Behörden festgelegten Reagenzien in den Anwendungsbereich dieses Dokuments. Nichtsdestotrotz können die Behörden oder andere interessierte Parteien im Bereich der Tiergesundheit sich dazu entschließen, in bestimmten und eng umrissenen Situationen, beispielsweise bei neu aufkommenden, exotischen oder seltenen Krankheiten, davon abzuweichen.
Dieses Dokument ist nicht auf alle vorhandenen diagnostischen Reagenzien anwendbar, insbesondere nicht auf solche, für die bestimmte in diesem Dokument beschriebene Parameter nicht in Übereinstimmung mit internationalen Anforderungen gültig bewertet werden können, z. B. weil kein spezifisches Referenznormal und/oder keine zugänglichen und ordnungsgemäß validierten Referenzmaterialien zur Verfügung stehen.
Die PCR-Diagnostik umfasst üblicherweise die Anwendung einer Nukleinsäure-Extraktion und/oder eines Reinigungsreagenzes und eines PCR-Reagenzes. Das PCR-Verfahren (sofern anwendbar) umfasst die aufeinanderfolgende Verwendung dieser verschiedenen Reagenzien. Die Prüfung des PCR-Reagenzes kann durchgeführt werden, wenn der Antragsteller einen Nachweis der Validität des PCR-Reagenzes für die Verwendung in diagnostischen Untersuchungen im Bereich der Tiergesundheit vorlegt, indem seine diagnostischen Leistungsmerkmale bei Verwendung mit Nukleinsäure-Extrakten, die aus den in der Gebrauchsinformation beschriebenen verschiedenen Matrices erhalten wurden, nachgewiesen werden. Die Prüfung eines vollständigen PCR-Verfahrens durch den Antragsteller und die Prüfstelle wird nur durchgeführt, wenn das PCR-Reagenz nicht von Nukleinsäure-Extraktions- und/oder -Reinigungssystemen gesondert geprüft werden kann. Die alleinige Prüfung der Reagenzien für Nukleinsäure-Extraktion und/oder -Reinigung wird in diesem Dokument nicht behandelt.
In diesem Dokument wird nicht der Schritt der Verifizierung eines Reagenzes durch den Anwender behandelt (Annahme eines Analyseverfahrens).
ANMERKUNG Durch Prionen verursachte Krankheiten sind nicht im Anwendungsbereich dieses dritten Teils der Normenreihe EN 18000 enthalten. Im Unterschied zu anderen Infektionskrankheiten werden durch Prionen verursachte Krankheiten nicht mithilfe von PCR-Tests diagnostiziert, weil Prionen ein Nukleinsäure-Bestandteil fehlt und sie lediglich aus einem anormal gefalteten Konformer des normalen Wirtsproteins bestehen.
Analyses de diagnostic en santé animale - Contrôle des réactifs de diagnostic in vitro - Partie 3 : Réactifs pour les techniques PCR
Le présent document spécifie le contrôle et l’approbation des réactifs de diagnostic in vitro utilisés en santé animale pour la détection, et/ou la quantification absolue d’acide nucléique spécifique à un pathogène (ADN ou ARN) par PCR (par exemple, PCR en point final, PCR en temps réel, PCR à transcription inverse).
Le présent document s’applique aux réactifs de diagnostic, en priorité pour les maladies infectieuses (dues à des bactéries, des virus, des champignons ou des parasites, y compris des marqueurs génétiques associés à une pathogénicité telle qu’une résistance antimicrobienne ou la production de toxines) et les espèces animales associées pour lesquelles une harmonisation des pratiques dans ce domaine est nécessaire, c’est-à-dire celles pour lesquelles le cadre réglementaire national, régional ou international prévoit le contrôle des échanges d’animaux et/ou de produits animaux et/ou la définition d’un état sanitaire (absence d’infection) des zones, établissements ou individus. Quoi qu’il en soit, tous les réactifs désignés par les autorités compétentes relèvent du domaine d’application du présent document. Néanmoins, les autorités ou toute autre partie prenante de la santé animale peuvent choisir de déroger à ces exigences dans des situations spécifiques et très limitées, telles que des maladies émergentes, exotiques ou rares.
Le présent document ne s’applique pas à tous les réactifs de diagnostic existants, en particulier ceux pour lesquels certains paramètres décrits dans le présent document ne peuvent pas être évalués de manière valable conformément aux exigences internationales en raison, par exemple, de l’absence d’une norme de référence spécifique et/ou de matériaux de référence accessibles et dûment validés.
Le diagnostic par PCR implique généralement l’utilisation d’un réactif d’extraction de l’acide nucléique et/ou de purification de l’acide nucléique, et d’un réactif de PCR. La méthode de PCR (lorsqu’elle est applicable) implique l’utilisation successive de ces différents réactifs. Le contrôle des réactifs de PCR peut être réalisé si le demandeur fournit la preuve de la validité du réactif de PCR pour utilisation dans le cadre de l’analyse diagnostique en santé animale, en mettant à disposition ses performances de diagnostic avec les extraits d’acide nucléique obtenus à partir des différentes matrices décrites dans la notice d’utilisation. Le contrôle d’une méthode de PCR complète par le demandeur et l’organisme de contrôle est réalisé uniquement si le réactif de PCR est indissociable des systèmes d’extraction de l’acide nucléique et/ou de purification de l’acide nucléique. Le présent document ne couvre pas le contrôle des réactifs d’extraction de l’acide nucléique et/ou de purification de l’acide nucléique uniquement.
Le présent document ne couvre pas l’étape de vérification d’un réactif par l’utilisateur (adoption de la méthode d’analyse).
NOTE Les maladies à prions ne relèvent pas du domaine d’application de la présente troisième partie de la série EN 18000. Contrairement à d’autres maladies infectieuses, les maladies à prions ne sont pas diagnostiquées à l’aide d’analyses par PCR, car les prions ne contiennent pas d’acide nucléique et se composent uniquement d’un conformère anormalement replié de la protéine hôte normale.
Diagnostične analize zdravja živali - Nadzor diagnostičnih reagentov in vitro - 3. del: Reagenti za metode PCR
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-junij-2025
Diagnostične analize zdravja živali - Nadzor diagnostičnih reagentov in vitro - 3.
del: Reagenti za metode PCR
Animal health diagnostic analyses - Control of in vitro diagnostic reagents - Part 3:
Reagents for PCR techniques
Tiergesundheitsdiagnostische Analysen - Kontrolle von in-vitro-diagnostischen
Reagenzien - Teil 3: Reagenzien für PCR Verfahren
Analyses de diagnostic en santé animale - Contrôle des réactifs de diagnostic in vitro -
Partie 3 : Réactifs pour les techniques PCR
Ta slovenski standard je istoveten z: prEN 18000-3
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
11.220 Veterinarstvo Veterinary medicine
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
DRAFT
EUROPEAN STANDARD
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2025
ICS 11.220
English Version
Animal health diagnostic analyses - Control of in vitro
diagnostic reagents - Part 3: Reagents for PCR techniques
Analyses de diagnostic en santé animale - Contrôle des Tiergesundheitsdiagnostische Analysen - Kontrolle von
réactifs de diagnostic in vitro - Partie 3 : Réactifs pour in-vitro-diagnostischen Reagenzien - Teil 3:
les techniques PCR Reagenzien für PCR Verfahren
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 469.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
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, Türkiye 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 European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18000-3:2025 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 General control steps . 10
5 Prerequisites of the PCR reagent control for the control organisation . 10
5.1 General . 10
5.2 Reference material . 10
5.3 Définition of the purpose of the PCR reagents and of the PCR method when applicable . 11
5.4 Additional useful information . 11
6 Initial conformity control . 11
6.1 General . 11
6.2 Characterization of the reagents by the applicant and documentary review by the
control organization . 11
6.2.1 General . 11
6.2.2 Définition of the interpretation method and threshold(s) . 14
6.2.3 Analytical spécificity of the PCR reagent . 14
6.2.4 Analytical sensitivity of the PCR reagent (LOD and MDL ) . 15
PCR PCR
6.2.5 Operating range of the quantitative PCR reagent (linearity, amplification éfficiéncy and
limit of quantification) when applicable . 15
6.2.6 Within-laboratory reproducibility of the PCR reagent . 16
6.2.7 Analytical sensitivity of the PCR method (MDL ) when applicable . 16
METHOD
6.2.8 Accuracy of the quantitative PCR method (operating range and limits of quantification)
when applicable . 16
6.2.9 Within-laboratory reproducibility of the PCR method when applicable . 17
6.2.10 Interlaboratory reproducibility of the PCR method when applicable . 17
6.2.11 Diagnostic sensitivity and diagnostic spécificity . 17
6.2.12 Validation of the conditions of use - Robustness . 17
6.2.13 Vérification of the stability . 17
6.3 Initial control of the reagents by the control organisation . 18
6.3.1 General . 18
6.3.2 Analytical spécificity of the PCR reagent . 18
6.3.3 Analytical sensitivity of the PCR reagent (MDL ) . 19
PCR
6.3.4 Operating range of the quantitative real-time PCR reagent (linearity, amplification
éfficiéncy and limit of quantification) . 19
6.3.5 Analytical sensitivity of the PCR method (MDL ), when applicable . 19
METHOD
6.3.6 Diagnostic sensitivity and spécificity of the PCR method when applicable . 19
6.3.7 Accuracy of the quantitative PCR method (validity range and limit of quantification),
when applicable . 19
7 Batch-to-batch control . 19
7.1 Control at the start of the batch shelf-life . 19
7.2 Control during the batch shelf-life . 20
7.3 Derogations from systematic batch-to-batch control . 20
8 Special cases . 20
8.1 Multiple protocols . 20
8.2 Multiple matrices . 20
8.3 Pooling of samples . 20
Bibliography . 22
European foreword
This document (prEN 18000-3:2025) has been prepared by Technical Committee CEN/TC "Animal
health diagnostic analyses", the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
Introduction
The purpose of the EN 18000 series is to facilitate the mutual recognition of the work of the animal
health in vitro diagnostic reagent control organisations at European level (or even more widely) and
thus to eventually allow the use of strategic reagents controlled by a single third-party control
organization for a given disease.
The EN 18000 series establishes the requirements for the control of in vitro diagnostic reagents in animal
health. This series is divided into three parts.
The first part concerns terms and définitions, and the submission of a reagent dossier to a control
organization for control and approval.
The second part concerns the spécific aspects of the control by such organizations of an immunological
diagnostic reagent.
The third part concerns the spécific aspects of the control by such organizations of a polymerase-chain
reaction (PCR) diagnostic reagent for the detection or quantification of pathogén-spécific nucleic acid. It
involves control organizations (CO) and applicants (including their subcontractors, when relevant).
Like any standard, this document is intended to be voluntary and, if its use is prescribed by a competent
authority or any other animal health stakeholder, it will be up to them to determine for which diseases
and to which extent this document will be applied by the control bodies they have designated for this
purpose.
1 Scope
This document spécifiés the control and approval of in vitro diagnostic reagents used in animal health
for the detection, and/or absolute quantification of pathogén-spécific nucleic acid (DNA or RNA) by PCR
(e.g. endpoint PCR, real-time PCR, reverse transcription-PCR).
This document is applicable to diagnostic reagents as a priority for infectious diseases (due to bacteria,
viruses, fungi, or parasites, including genetic markers associated with pathogenicity, such as
antimicrobial resistance or toxin production) and associated animal species for which harmonization
of practices in this area is needed, i.e. those for which the national, regional or international regulatory
framework provides for the control of trade in animals and/or animal products and/or the définition
of a health status (absence of infection) of areas, establishments or individuals. Anyhow, all reagents
designated by the competent authorities fall under the scope of this document. Nevertheless, the
authorities or any other animal health stakeholder can choose to derogate in spécific and very limited
situations such as emerging, exotic or rare diseases.
This document is not applicable to all existing diagnostic reagents, in particular those for which certain
parameters described in this document cannot be validly evaluated in accordance with international
requirements, due, e.g. to the absence of a spécific reference standard and/or accessible and duly
validated reference materials.
The PCR diagnosis usually involves the use of a nucleic acid extraction and/or purification reagent, and
a PCR reagent. The PCR method (when applicable) involves the successive use of these distinct
reagents. PCR reagent control can be performed if the applicant provides evidence of the validity of the
PCR reagent for use in the animal health diagnostic analysis, by proving its diagnostic performances
with nucleic acid extracts obtained from the different matrices described in the instruction for use. The
control of a complete PCR method by the applicant and the control organization is performed only if the
PCR reagent cannot be dissociated from an nucleic acid extraction and/or purification systems. This
document does not cover the control of the nucleic acid extraction and/or purification reagents, only.
This document does not cover the step in which the user vérifiés a reagent (analysis method adoption).
NOTE Prion diseases are not included in the scope of this third part of the EN 18000 series. Unlike other
infectious diseases, prion diseases are not diagnosed using PCR assays because prions lack a nucleic acid
component and consist solely of an abnormally folded conformer of the normal host protein.
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.
prEN 18000-1:2025, Animal health diagnostic analyses - Control of in vitro diagnostic reagents - Part 1:
Application file for the initial and the batch-to-batch control
prEN ISO 22174:2024, Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection
and quantification of microorganisms - General requirements and definitions (ISO/DIS 22174:2022)
3 Terms and definitions
For the purposes of this document, the terms and définitions given in prEN 18000-1:2025 and the
following apply.
NOTE These terms are written in italics throughout the EN 18000 series.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
absolute quantification by real-time PCR
procedure involving PCR amplification to determine the concentration of a target nucleic acid in a sample
by comparison with a standard curve, derived from standards containing a définéd amount of target
[SOURCE: prEN ISO 22174:2024]
3.2
accepted reference value
value that serves as an agreed-upon reference for comparison, and which is derived as:
a) theoretical or established value, based on sciéntific principles;
b) an assigned or cértifiéd value, based on experimental work of some national or international
organization;
c) a consensus or cértifiéd value, based on collaborative experimental work under the auspices of a
sciéntific or engineering group;
d) when a), b) and c) are not available, the expectation of the (measurable) quantity, i.e. the mean of
a spécifiéd population of measurement
[SOURCE: [1]]
3.3
accuracy
closeness of agreement between the test result and the accepted reference value
Note 1 to entry: The term accuracy, when applied to a set of test results, involves a combination of random
components and a common systematic error or bias component.
[SOURCE: [1]]
3.4
background fluorescence
intrinsic level of fluoréscéncé resulting from the reagents, consumables and instruments used
[SOURCE: [2]]
3.5
bias
difference between the expectation of the test results and an accepted reference value
Note 1 to entry: Bias is the total systematic error as contrasted to random error. There may be one or more
systematic error components contributing to the bias. A larger systematic difference from the accepted reference
value is réfléctéd by a larger bias value.
[SOURCE: [1]]
3.6
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)
form
[SOURCE: [2]]
3.7
probe
nucleic acid molecule with a définéd sequence used to detect target DNA by a hybridization-mediated
reporter reaction
3.8
detection
recognition of the presence of the nucleic acid target
[SOURCE: [2]]
3.9
endpoint PCR
procedure using PCR amplification followed by separate detection of PCR products after the completion
of the PCR
[SOURCE: [2], modifiéd - "amplicons" has been removed and replaced by "PCR products".]
3.10
external amplification control
control nucleic acid added to an aliquot of the extracted nucleic acid in a définéd amount or copy number
serving as a control for amplification in a separate reaction
Note 1 to entry: It is strongly recommended that the external amplification control PCR product be distinguishable
from the target PCR product (e.g. by insertion of a restriction enzyme target sequence).
[SOURCE: [2], modifiéd - "amplicon" has been removed and replaced by "PCR product".]
3.11
internal amplification control
nucleic acid added to each reaction in a définéd amount or copy number which serves as an internal
control for amplification
Note 1 to entry: This nucleic acid sequence can be endogeneous (naturally present in the tested matrix) or
exogeneous (naturally absent in the tested matrix).
Note 2 to entry: An exogenous internal amplification control can be homologous (amplifiéd using the same primers
as used for amplification of the target) or heterologous (amplifiéd using different primers than those used for
amplification of the target). An homologous internal amplification control PCR product shall be distinguishable
from the microbial target PCR product (e.g. by size, or by insertion of a different probe-binding sequence).
[SOURCE: [2], modifiéd - "amplicon" has been removed and replaced by "PCR product".]
3.12
multiplex PCR
PCR allowing the detection of multiple targets simultaneously within a single reaction tube, where more
primer pairs (and probe) are used within one master mix
[SOURCE: [2]]
3.13
nucleic acid
polymer of DNA or RNA
[SOURCE: [2]]
3.14
nucleic acid extraction
sample treatment for the release of target nucleic acids
[SOURCE: [2]]
3.15
nucleic acid purification
method to reduce the amount of PCR inhibitors in the eluate
[SOURCE: [2]]
3.16
polymerase chain reaction
PCR
enzymatic procedure that allows in vitro amplification of DNA
[SOURCE: [2]]
3.17
PCR method
diagnostic process allowing the detection, the relative quantification, and/or the absolute quantification
of a targeted nucleic acid by PCR or real-time PCR from a sample
Note 1 to entry: The diagnostic process generally involves the sample preparation, the nucleic acid extraction, the
nucleic acid purification, the nucleic acid amplification by PCR (possibly performed after the reverse transcription
of RNA into DNA), the PCR product detection, and the interpretation of the PCR signal.
Note 2 to entry: For in vitro reagents in animal health, the amplifiéd nucleic acid is a pathogen sequence and the
sample can include animal or environmental sample.
3.18
PCR reagent
reagent necessary for the PCR
Note 1 to entry: Reminder: the term reagent is définéd in part 1 [3]
3.19
primer
oligonucleotide of définéd length and sequence complementary to a segment of the target nucleic acid
sequence, used to signal the starting point for DNA polymerase to extend the new DNA strand
[SOURCE: [2]]
3.20
quantification cycle
Cq
real-time PCR cycle at which the fluoréscéncé signal can be distinguished from the background
fluorescence entering into the exponential phase of target amplification
Note 1 to entry: Quantification cycle is a generic term which includes cycle threshold (C ), crossing point (C ), take
t p
off point (TOP) and all other instrument spécific terms referring to the fractional cycle used to detect or quantify
the target in the real-time PCR assay.
Note 2 to entry: The quantification cycle is based either on a threshold applied to all samples or on a regression
analysis of the signal, for each sample.
[SOURCE: [2]]
3.21
quantitative PCR
method allowing the quantification in a nucleic acid template of the number of a spécific nucleic acid
sequence using spécific oligonucleotides
[SOURCE: [2]]
3.22
real-time PCR
procedure which combines PCR amplification with the detection and/or quantification of spécific PCR
products during the amplification process
[SOURCE: [2], modifiéd - "amplicons" has been removed and replaced by "PCR products".]
3.23
relative quantification by real-time PCR
procedure involving PCR amplification to determine the levels of changes of a target nucleic acid relative
to the levels of a reference nucleic acid of known concentration, measured within the same or in separate
PCR reactions
[SOURCE: [2]]
3.24
reverse transcription
RT
synthesis of complementary single-stranded DNA (cDNA) from an RNA template using a reverse
transcriptase
[SOURCE: [2]]
3.25
reverse transcription-PCR
RT-PCR
method consisting of two reactions, a reverse transcription (RT) of RNA to single-stranded
complementary DNA (cDNA), followed by a PCR
Note 1 to entry: A one step RT -PCR is performed in a single tube.
Note 2 to entry: A two step RT -PCR can be either performed sequentially in a single tube or in two different tubes.
[SOURCE: [2]]
3.26
ribonucleic acid
RNA
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: [2]]
3.27
target nucleic acid sequence
nucleic acid sequence selected for amplification
[SOURCE: [2]]
3.28
thermal cycler
automatic device that performs définéd heating and cooling cycles necessary for PCR or real-time PCR
Note 1 to entry: The thermal cycler can be a block-based or (individual) reaction-chamber-based thermal cycler.
[SOURCE: [4]]
3.29
trueness
closeness of agreement between the average value obtained from a large series of test results and an
accepted reference value
Note 1 to entry: The measure of trueness is usually expressed in terms of bias.
[SOURCE: [1]]
4 General control steps
The reagents are controlled in accordance with this document when so required. Depending on the
context (e.g. regulation, disease control programme, etc.) of the implementation of analyses with such
reagents, the control can include an initial conformity control and several batch-to-batch controls.
Certain steps of the PCR reagent control can be subcontracted by the CO, provided that it ensures that
the subcontractors possess the necessary skills (e.g. accreditation [5]) and are independent. This
accreditation shall include the same technique and disease as those covered by the PCR reagents control.
Commitment of impartiality and confidéntiality shall be guaranteed by written disclosure of potential
conflicts of interest in accordance with Annex C of prEN 18000-1:2025.
Subcontracted laboratories shall work following purposes and protocols approved by the CO. The list
of subcontracted laboratories shall be public.
This document describes the requirements for each of the steps of the control, without défining the
criteria of conformity, which shall be spécifiéd beforehand by the CO according to the state of the art
(e.g. existing disease tést-spécific standards, consensus conference recommendations, sciéntific
literature). If necessary, or based on a particular risk or epidemiological context, additional controls can
be performed that are not described in detail in this standard.
5 Prerequisites of the PCR reagent control for the control organisation
5.1 General
The prerequisites for the control of reagents include:
— the availability of reference materials (RMs);
— the définition of the minimum performance requirements of the PCR reagents or when applicable
of the PCR method (e.g. regulations, standards, guidelines);
— additional useful information (e.g. templates) provided by the CO, when relevant to harmonise as
far as possible the implementation of control between the CO and the applicant.
5.2 Reference material
The CO shall possess characterised positive RMs and negative RMs. The RMs can exist in the form of
different matrices in different levels of concentration.
The CO shall provide the applicant with RMs ([6]), so that the applicant can prepare its internal RMs in
order to conduct the controls described in this document. With these RMs, it shall provide the
characteristics listed in prEN 18000-1:2025, Annex A. For cértifiéd RMs, the characteristics appearing
on the cértificaté to be provided are listed in [7].
The RMs can also be prepared and/or supplied by third parties, provided that they meet the criteria
définéd by the CO and have been approved by the latter.
Certain RMs are définéd by regulations or standards (e.g. WOAH Manuals of diagnostic tests [8][9],
Reference Laboratories guidelines).
The RMs can be prepared from naturally or artificially contaminated samples, or in the form of nucleic
acid. If possible, the CO shall provide the applicant with the production protocol.
The CO is not required to provide the fiéld samples or the samples produced by experimental infection
that are necessary for the assessment of the diagnostic sensitivity and specificity. However, the CO shall
recommend the method(s) and/or procedure(s) used for sample characterization.
NOTE If a user of the diagnostic method chooses an extraction method different from the applicant or the CO
extraction method, it is its own responsibility to ensure that the needed RMs are available to generate appropriate
verification data for method performance.
5.3 Definition of the purpose of the PCR reagents and of the PCR method when
applicable
The CO shall specify in writing the purpose of the reagents for the applicant including regulatory
and/or particular requirements, such as the determination of a minimum detection level, the modalities
of use of the reagents, the animal species and matrices concerned, etc. It shall inform the applicant of
the details of its control protocol (initial conformity control and batch-to-batch control). All above-
mentioned purposes, requirements and the protocols for the reagent control shall also be made public.
5.4 Additional useful information
The applicant shall specify the equipment (e.g. devices, instruments, thermal cycler) and reagents (other
than PCR reagent; e.g. nucleic acid extraction and purification systems) used for this characterization.
The applicant shall describe if spécific nucleic acid extraction and purification systems should be avoided.
The cost implications of this design for the CO (workload and control costs) and/or the applicant (reagent
amount) shall also be taken into account.
6 Initial conformity control
6.1 General
This clause and the following describe the respective responsibilities of the applicant (6.2) and the CO
(6.3) with regard to the initial conformity control and the batch-to-batch control respectively (Clause
7). The following Table 1 (and Table 2, Table 3 or Table 4 when applicable) summarizes these
obligations.
6.2 Characterization of the reagents by the applicant and documentary review by the
control organization
6.2.1 General
After developing a reagent, the applicant shall proceed with the characterization of its performance for
a spécifiéd protocol, based on one or more production batches.
The applicant shall send a filé to the CO. This filé shall contain an administrative part, describing the
manufacturer, the applicant and the reagent(s), and a technical part (initial technical dossier), including
the information listed in prEN 18000-1:2025, 5.2, 5.3 and Annex B, respectively. In the technical part,
the PCR reagent or the PCR method characterization (when applicable) shall take account of the
parameters listed in Table 1 (and in Table 2, Table 3 or Table 4 when applicable) and described below.
All samples used by the applicant for the characterization of the various required parameters, except
for RMs provided by the CO, shall be described by the applicant as precisely as possible (within the limits
of confidéntiality requirements). In particular, it shall be spécifiéd whether these samples are naturally
contaminated or the result of laboratory spiking (e.g. with pathogen or nucleic acid). Similarly, the animal
species of origin, the infectious and/or vaccinal status of the animals, and the matrix(ces) concerned
shall be spécifiéd. In addition, if relevant, other characteristics such as the geographical region of origin
of the animals, or even their sex and age, can be requested by the CO.
The CO shall have access to the applicant’s raw data, whenever necessary, except data pertaining to
trade secrecy. The CO shall protect the confidéntiality of the information in the filé.
The CO reviews the filé. If it meets the requirements, the initial control can be performed (see 6.3).
Table 1 — Criteria assessed in the control of qualitative PCR reagents and responsibilities
Initial conformity control Batch-to-batch control
Assessment of the performance parameters Fol
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