CERTIFICATION REPORT A set of three plasmid DNA calibration solutions bearing a porcine-specific DNA fragment
Certified Reference Materials: ERM®-AD483a, ERM®-AD483b, ERM®-AD483c
This report describes the preparation and characterisation of a set of plasmid solutions, ERM-AD483a, ERM-AD483b and ERM-AD483c. The materials were produced in accordance with ISO Guide 34:2009 [1].
A DNA fragment specific for the identification of a porcine target was cloned into a pUC18 vector to construct the pIRMM-0104 plasmid. The nucleic acid sequence of the entire pIRMM-0104 plasmid was determined by dye terminator cycle sequencing. The plasmid was eluted in TElow buffer, and its concentration was measured by ultraviolet (UV) spectrophotometry. Afterwards, it was gravimetrically diluted to three different concentration levels. The plasmid copy number concentration of the three concentration levels were certified by digital and droplet digital quantitative polymerase chain reaction methods, dPCR and ddPCR respectively. In addition, between-unit homogeneity, as well as short-term and long-term stability was assessed in accordance with ISO Guide 35:2006 [2].
The materials are intended for the determination of a cut-off value to discriminate positive samples (containing the porcine target sequence) from negative samples by quantitative PCR as defined in the Standard Operating Procedure of the EU Reference Laboratory for Animal proteins (EURL-AP) according to Commission Regulation (EU) No 51/2013 [3, 4].
As any certified reference material (CRM), ERM-AD483 can also be used for control charts or validation studies. The CRM is available as a set of three vials, each containing at least 1 mL of plasmid solution. The minimum amount of sample to be used is 4 µL.
MATRAI Janka;
CORBISIER Philippe;
MAZOUA Stephane;
KORTEKAAS Anna Maria;
CHUNG Pui Yan Jenny;
TRAPMANN Stefanie;
EMONS Hendrik;
2015-12-16
Publications Office of the European Union
JRC97915
978-92-79-52306-9,
1831-9424,
EUR 27505,
OP LA-NA-27505-EN-N,
https://publications.jrc.ec.europa.eu/repository/handle/JRC97915,
10.2787/9570,
Additional supporting files
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