Method of reference and SOPs

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 :
[NEW] Ruminant calibrant
Porcine calibrant
Poultry calibrant

List of approved master mixes

Product name Reference
(may vary depending on the packaging)		 Producer PCR test
Ruminant Pig Poultry
Brilliant II QPCR Low Rox Master mix 600806 Agilent V V V
RealQ Plus Master mix for probe A314499 Ampliqon V 🔍 🗓
Takyon™ Low Rox Probe MasterMix dTTP Blue UF-LPMT-B0705 Eurogentec V 🔍 🗓
qPCR™ Mastermix Plus without UNG RT-QP2X-03-WOU+ Eurogentec X ⏳ ⏳
TaqMan® Universal PCR Master Mix 4304437 Applied Biosystems by Life technologies X 🔍 🗓
PerfeCta® qPCR ToughMix® 95112-250 Quantabio X 🔍 🗓
Xpert Fast PROBE Mastermix (Uni) GE30.5100 GRISP research solutions X 🔍 🗓
SensiFAST Probe No-ROX kit BIO-86005 Meridian Bioscience X 🔍 🗓
Legend : V = recommended for this PCR test, X = not recommended for this PCR test,
⏳ = temporarily recommended, 🔍 = under evaluation, 🗓 = not yet evaluated

Note: The thermal cycling program must be performed in accordance with the supplier’s guidelines, except for the annealing and elongation temperature, which is specified in the PCR SOP.

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 specificity of the ruminant PCR test was experimentally tested with Brilliant II QPCR Low Rox Master mix (Agilent, Santa Clara, CA, USA), RealQ Plus Master mix for probe (Ampliqon A/S, Odense, Denmark) and Takyon™ Low Rox Probe MasterMix dTTP Blue (Eurogentec, Seraing, Belgium) successfully on a collection of reference DNAs: five ruminant species were all detected (cattle – Bos taurus, sheep – Ovis aries, goat – Capra hircus, stag or red deer – Cervus elaphus and roe deer – Capreolus capreolus), while non-target species are not detected and this was tested on seven terrestrial mammalian species (pig – Sus scrofa domesticus, wild boar – Sus scrofa scrofa, horse – Equus caballus, donkey – Equus asinus, hare – Lepus europaeus, rat – Rattus rattus and human – Homo sapiens), four sea mammals (striped dolphin – Stenella coeruleoalba, Risso’s dolphin – Grampus griseus, Harbor porpoise – Phocoena phocoena, seals – Phocidae), nine bird species (chicken – Gallus gallus, turkey – Meleagris gallopavo, duck – Cairina moschata, guinea fowl – Numida meleagris, goose – Anser spp., quail – Coturnix japonica, pheasant – Phasianus colchicus, pigeon – Columba livia and ostrich – Struthio camelus), 14 fish species (Gadus morhua, Micromesistius poutassou, Sebastes spp., Mallotus villosus, Scomber scombrus, Clupea harengus, Merluccius merluccius, Sardina pilchardus, Engraulis encrasicolus, Trisopterus esmarki, sand eel, Sprattus sprattus, Salmo salar, Raja spp.), six crustacean species (red king crab – Paralithodes camtschaticus, Antartic krill – Euphausia superba, Common shrimp – Crangon crangon, Langoustine – Nephrops norvegicus, Whiteleg shrimp – Penaeus vannamei, European lobster – Homarus gammarus), one mollusk (squid – Teuthida sp.), and seven plant samples (soybean – Glycine max, maize – Zea mays, rapeseed – Brassica napus, wheat – Triticum aestivum, rice – Oryza sativa, tomato – Solanum lycopersicum, sugar beet – Beta vulgaris). As observed with the master mixes used during PCR test validation, non-specific signals were observed with two sea mammals belonging to the order Cetacea (Tursiops truncatus and Ziphius cavirostris). However, with regard to the intended purpose of the analysis, this does not represent a practical issue.

    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
    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