Horse DNA detection method

• Detection of horse DNA using real-time PCR
• SOP on use of reference material with respect to recommendation 2014/180/EU
• Labs for confirmatory testing in each Member State

Method of reference for the detection of animal proteins in feed

The Annex VI of Commission Regulation n° 152/2009 was entirely revised and amended by Commission Regulation n° 51/2013 published on the 16th of January 2013

The new regulation relies on the combination of light microscopy and real time PCR for the detection of animal proteins in feed in the European Union. This regulation also relies on Standard Operating Procedures (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.

• Commission Regulation n°51/2013
  amending Regulation (EC) No 152/2009 as regards the methods of analysis for the determination of constituents of animal origin for the official control of feed
• Commission Regulation n°152/2009
  laying down the methods of sampling and analysis for the official control of feed
• TNO Triskelion bv ruminant PCR test (including sequences)

Calibration kits for ruminant and pig detection by PCR

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

For ordering and information, go to the following webpages :
https://crm.jrc.ec.europa.eu/p/erm-AD482
https://crm.jrc.ec.europa.eu/p/erm-AD483

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   

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. Minor revisions are labelled V.1.# and major revisions are labelled V.#.0. 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	NEW
EURL-AP SOP poultry PCR	V1.0	BINDING	NEW
EURL-AP SOP cut-off determination	V2.0	BINDING
EURL-AP SOP operational schemes	V5.0	BINDING	NEW
EURL-AP SOP observation flowchart	V1.0	BINDING
EURL-AP SOP slide mounting	V1.0	BINDING
EURL-AP SOP staining methods	V1.0	OPTIONAL

Test – JH – Other tables

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