Certification of plasmid DNA copy number concentrations bearing a ruminant-specific DNA fragment in three buffered solutions: ERM®-AD482ka, ERM®-AD482kb, ERM®-AD482kc
ERM®-AD482k is a set of three certified reference materials (CRMs) produced within the scope of ISO 17034:2016 [1] accreditation.
The European Union Reference Laboratory for Animal proteins in feedingstuffs (EURL-AP) inserted a specific sequence for ruminant identification into the pUC18 plasmid and cloned it into E. coli. The plasmid was multiplied, purified, linearised, quantified by UV spectrophotometry, sequenced by Sanger sequencing, and then gravimetrically diluted to three target concentrations, with copy numbers certified by digital PCR. The CRM is available as a set of three polypropylene vials containing at least 1 mL of solution at one of the three copy number concentrations.
Between-unit homogeneity was quantified and stability during transport and storage was assessed in accordance with ISO 33405:2024 [2].
The material was characterised by an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025:2017 [3]. Technically invalid results were removed but no outlier was eliminated unless a technical reason for the deviation was found.
Uncertainties of the certified values include uncertainties related to possible inhomogeneity, instability and characterisation.
The material is intended to be used in quantitative PCR (qPCR), for the determination of the cut-off value differentiating positive samples (containing the ruminant target sequence) from negative samples.
Before release of the set of the three CRMs, the certification project was subjected to peer-review involving both internal and external experts.
MAZOUA Stephane;
MEEUS Nele;
KHALED GIJON Mohamed;
TRAPMANN Stefanie;
2025-05-05
Publications Office of the European Union
JRC141444
978-92-68-25148-5 (online),
OP KJ-01-25-144-EN-N (online),
https://publications.jrc.ec.europa.eu/repository/handle/JRC141444,
10.2760/0953383 (online),
Additional supporting files
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