Method of reference for the detection of animal proteins in feed

Annex VI of Commission Regulation n° 152/2009 was revised and amended by Commission Regulation n° 51/2013, by Commission Implementation Regulation n° 1560/2020 and recently by Commission implementing Regulation (EU) 2022/893. The new regulation relies on the combination of light microscopy and real time PCR for the detection of animal proteins in feed and on Standard Operating Protocols (SOPs) edited by the EURL-AP for technical implementation.

These SOPs are binding or optional complements to the regulation and are available for download on this page.

Calibration kits for ruminant, pig and poultry detection by PCR

The calibration kits ERM-AD482 RUMINANT pDNA CALIBRANT, ERM-AD483 PORCINE pDNA CALIBRANT and ERM-AD484k POULTRY pDNA CALIBRANT can be ordered from JRC Geel. They are now Certified European Reference Materials.

For ordering and information, go to the following webpages :
Ruminant calibrant
Porcine calibrant
Poultry calibrant

List of approved master mixes

Detection of ruminant DNA

As mentioned in the  Commission Regulation (EU) No 51/2013 as well as in the SOP “ Detection of ruminant DNA in feed using real-time PCR”, it is crucial to be careful that the master mix used is fit for the purpose and does not contain bovine serum albumin (BSA) or other reagents susceptible to lead to false results due to presence of animal DNA.

The EURL-AP recommends to use one of the following mastermixes:

  • “Universal mastermix” reference number DMML-D2-D600 (Diagenode, Liège, Belgium) www.diagenode.com
  • “qPCR MasterMix” reference number RT-QP2X-03 (Eurogentec, Seraing, Belgium) www.eurogentec.com   
    Quote on demand at isales@eurogentec.com
  • Brilliant II QPCR ROX Master Mix www.agilent.com
    “Brilliant II QPCR Low ROX Master Mix” reference number 600806 (Agilent, Santa Clara, CA, USA)
    “Brilliant II QPCR High ROX Master Mix” reference number 600805 (Agilent, Santa Clara, CA, USA)

List of SOPs for download

This list is updated and contains only valid versions.  The binding or optional character of a SOP is indicated.  The original version is labelled V.1.0. Revisions are labelled V.#.#. The period of validity of the different versions are mentioned at the end of the document.

SOPs recently uploaded are tagged NEW for two months after publication date.

Title Version
EURL-AP SOP DNA extraction V1.1 BINDING
EURL-AP SOP ruminant PCR V1.3 BINDING
EURL-AP SOP pig PCR V1.0 BINDING
EURL-AP SOP poultry PCR V1.2 BINDING NEW 
EURL-AP SOP cut-off determination – ruminant V2.0 BINDING
EURL-AP SOP cut-off determination – pig V1.0 BINDING
EURL-AP SOP cut-off determination – poultry V1.0 BINDING
EURL-AP SOP operational schemes V5.1 BINDING
EURL-AP SOP observation flowchart V2.0 BINDING
EURL-AP SOP slide mounting V1.0 BINDING
EURL-AP SOP staining methods V1.0 OPTIONAL