# Commission Regulation (EU) No 51/2013Show full title

Commission Regulation (EU) No 51/2013 of 16 January 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 (Text with EEA relevance)         

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U.K.

# Commission Regulation (EU) No 51/2013                     

# of 16 January 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                     

# (Text with EEA relevance)

THE EUROPEAN COMMISSION,

Having regard to the Treaty on the Functioning of the European Union,

Having regard to Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls                           performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules(1), and in particular Article 11(4) thereof,                        

Whereas:

(1)Article 7(1) of Regulation (EC) No 999/2001 of the European Parliament and of the Council of 22 May 2001 laying down rules                              for the prevention, control and eradication of certain transmissible spongiform encephalopathies(2) provides that the feeding to ruminants of protein derived from animals is prohibited. That prohibition is extended to animals                              other than ruminants and restricted, as regards the feeding of those animals with products of animal origin, in accordance                              with Annex IV to that Regulation.

(2)Article 11(1) of Regulation (EC) No 1069/2009 of the European Parliament and of the Council of 21 October 2009 laying down                              health rules as regards animal by-products and derived products not intended for human consumption and repealing Regulation                              (EC) No 1774/2002(3) prohibits the feeding of terrestrial animals of a given species other than fur animals with processed animal protein derived                              from the bodies or parts of bodies of animals of the same species, as well as the feeding of farmed fish with processed animal                              protein derived from the bodies or parts of bodies of farmed fish of the same species.

(3)Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official                              control of feed(4) sets out in its Annex VI the methods of analysis for the determination of constituents of animal origin for the official                              control of feed. The microscopic method, which is currently the only method validated to detect the presence of animal proteins                              in feed, is able to distinguish the presence of constituents derived from terrestrial animals from the presence of constituents                              derived from fish, but unable to quantify with a sufficient level of accuracy the amount of animal constituents present in                              feed, and therefore should not be used for this purpose.

(4)A new method of detection of animal constituents based on polymerase chain reaction (PCR) was validated by the EU reference                              laboratory for animal proteins in feedingstuffs. An implementation study, organised with the national reference laboratories                              of the Member States, proved that the new method is sufficiently robust to be used as an official control method in the Union.                              This new method is able to detect the presence of animal constituents in feed, and also able to identify the species origin                              of these constituents. The use of this new method in combination with or in replacement of, as appropriate, the microscopic                              method would be very valuable for the control of the correct implementation of the feeding prohibitions laid down in Regulations                              (EC) No 999/2001 and (EC) No 1069/2009.

(5)Annex VI to Regulation (EC) No 152/2009 should therefore be replaced accordingly.

(6)The measures provided for in this Regulation are in accordance with the opinion of the Standing Committee on the Food Chain                              and Animal Health and neither the European Parliament nor the Council have opposed them,

HAS ADOPTED THIS REGULATION:

# Article 1U.K.

Annex VI to Regulation (EC) No 152/2009 is replaced by the text set out in the Annex to this Regulation.

# Article 2U.K.

This Regulation shall enter into force on the twentieth day following that of its publication in the *Official Journal of the European Union*.                  

This Regulation is binding in its entirety and directly applicable in all Member States.

## ANNEXU.K.

### ‘ANNEX VIU.K.**METHODS OF ANALYSIS FOR THE DETERMINATION OF CONSTITUENTS OF ANIMAL ORIGIN FOR THE OFFICIAL CONTROL OF FEED**

#### 1.PURPOSE AND SCOPE

The determination of constituents of animal origin in feed shall be performed by light microscopy or polymerase chain reaction                        (PCR) in accordance with the provisions laid down in this Annex.                  

These two methods make it possible to detect the presence of constituents of animal origin in feed materials and compound                        feed. However, they do not make it possible to calculate the amount of such constituents in feed materials and compound feed.                        Both methods have a limit of detection below 0,1 % (w/w).                  

The PCR method makes it possible to identify the taxonomic group of constituents of animal origin present in feed materials                        and compound feed.                  

These methods shall apply for the control of the application of the prohibitions laid down in Article 7(1) and Annex IV to                        Regulation (EC) No 999/2001 and in Article 11(1) of Regulation (EC) No 1069/2009.                  

Depending on the type of feed being tested, these methods may be used, within one single operational protocol, either on their                        own or combined together in accordance with the standard operating procedures (SOP) established by the EU reference laboratory                        for animal proteins in feedingstuffs (EURL-AP) and published on its website(5).                  

#### 2.METHODS

##### 2.1.**Light microscopy**

###### 2.1.1.*Principle*

The constituents of animal origin which may be present in feed materials and compound feed sent for analysis are identified                        on the basis of typical and microscopically identifiable characteristics like muscle fibres and other meat particles, cartilage,                        bones, horn, hair, bristles, blood, feathers, egg shells, fish bones and scales.                  

###### 2.1.2.*Reagents and equipment*

###### 2.1.2.1.Reagents

###### 2.1.2.1.1.Concentrating agent

###### 2.1.2.1.1.1.Tetrachloroethylene (specific gravity 1,62)

###### 2.1.2.1.2.Staining reagent

###### 2.1.2.1.2.1.Alizarin Red solution (dilute 2,5 ml 1M hydrochloric acid in 100 ml water and add 200 mg Alizarin Red to this solution)

###### 2.1.2.1.3.Mounting media

###### 2.1.2.1.3.1.Lye (NaOH 2,5 % w/v or KOH 2,5 % w/v)

###### 2.1.2.1.3.2.Glycerol (undiluted, viscosity: 1 490 cP)

###### 2.1.2.1.3.3.Norland ® Optical Adhesive 65 (viscosity: 1 200 cP) or a resin with equivalent properties for permanent slide preparation

###### 2.1.2.1.4.Mounting media with staining properties

###### 2.1.2.1.4.1.Lugol solution (dissolve 2 g potassium iodide in 100 ml water and add 1 g iodine while frequently shaking)

###### 2.1.2.1.4.2.Cystine reagent (2 g lead acetate, 10 g NaOH/100 ml water)

###### 2.1.2.1.4.3.Fehling’s reagent (prepared before use from equals parts (1/1) of two stock solutions A and B. Solution A: dissolve 6,9 g                           copper (II) sulphate pentahydrate in 100 ml water. Solution B: dissolve 34,6 g potassium sodium tartrate tetrahydrate and                           12 g NaOH in 100 ml water)

###### 2.1.2.1.4.4.Tetramethylbenzidine/Hydrogen peroxide. (dissolve 1 g 3,3’,5,5’ tetramethylbenzidine (TMB) in 100 ml glacial acetic acid and                           150 ml water. Before use, mix 4 parts of this TMB solution with 1 part 3 % hydrogen peroxide)

###### 2.1.2.1.5.Rinsing agents

###### 2.1.2.1.5.1.Ethanol ≥ 96 % (technical grade)

###### 2.1.2.1.5.2.Acetone (technical grade)

###### 2.1.2.1.6.Bleaching reagent

###### 2.1.2.1.6.1.Commercial sodium hypochlorite solution (9 - 14 % active chlorine)

###### 2.1.2.2.Equipment

###### 2.1.2.2.1.Analytical balance with an accuracy of 0,001 g

###### 2.1.2.2.2.Grinding equipment: mill - or mortar

###### 2.1.2.2.3.Sieves with square meshes of 0,25 mm and 1 mm width

###### 2.1.2.2.4.Conical glass separation funnel with a content of 250 ml with Teflon or ground glass stopcock at the base of the cone. Stopcock                           opening diameter shall be ≥ 4mm. Alternatively, a conical bottomed settling beaker may be used provided the laboratory has                           demonstrated that detection levels are equivalent to that obtained using the conical glass separation funnel.

###### **Separation funnel**

###### 2.1.2.2.5.Stereomicroscope covering at least a 6,5× to 40× final magnification range

###### 2.1.2.2.6.Compound microscope covering at least a 100× to 400× final magnification range with transmitted light bright field. Polarised                           light and differential interferential contrast can additionally be used

###### 2.1.2.2.7.Standard laboratory glassware

###### 2.1.2.2.8.Equipment for slide preparation: classical microscope slides, hollow slides, coverslips (20 × 20 mm), tweezers, fine spatula

###### 2.1.3.*Sampling and sample preparation*

###### 2.1.3.1.Sampling

A representative sample, taken in accordance with the provisions laid down in Annex I shall be used.                  

###### 2.1.3.2.Precautions to be taken

In order to avoid laboratory cross-contamination, all reusable equipment shall be carefully cleaned before use. Separation                        funnel pieces shall be disassembled before cleaning. Separation funnel pieces and glassware shall be pre-washed manually and                        then washed in a washing machine. Sieves shall be cleaned by using a brush with stiff synthetic hairs. A final cleaning of                        sieves with acetone and compressed air is recommended after sieving of fatty material like fishmeal.                  

###### 2.1.3.3.Preparation of samples other than fat or oil

###### 2.1.3.3.1.*Sample drying*: samples with a moisture content > 14 % shall be dried prior to handling.

###### 2.1.3.3.2.*Sample pre-sieving*: it is recommended to pre-sieve at 1 mm pelleted feeds and kernels and to subsequently prepare and analyse the two resulting                           fractions as distinct samples.

###### 2.1.3.3.3.*Sub-sampling and grinding*: at least 50 g of the sample shall be sub-sampled for analysis and subsequently ground.

###### 2.1.3.3.4.*Extraction and preparation of the sediment*: a portion of 10 g (accurate to 0,01 g) of the ground sub-sample shall be transferred into the separation funnel or conical                           bottomed settling beaker and 50 ml of tetrachloroethylene shall be added. The portion transferred into the funnel shall be                           limited to 3 g in case of fishmeal or other pure animal products, mineral ingredients or premixes which generate more than                           10 % of sediment. The mixture shall be vigorously shaken for at least 30 s and at least 50 ml more of tetrachloroethylene                           shall be added cautiously while washing down the inside surface of the funnel to remove any adhering particles. The resulting                           mixture shall be left to stand for at least 5 minutes before the sediment is separated off by opening the stopcock.

If a conical bottomed settling beaker is used then the mixture shall be vigorously stirred for at least 15 s and any particles                        adhering to the side of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean tetrachloroethylene.                        The mixture shall be left to stand for 3 minutes and then stirred again for 15 seconds and any particles adhering to the side                        of the beaker shall be carefully washed down the inside surface with at least 10 ml of clean tetrachloroethylene. The resulting                        mixture shall be left to stand for at least 5 minutes and then the liquid fraction is removed and discarded by careful decanting,                        taking care not to lose any of the sediment.                  

The sediment shall be dried and subsequently weighed (accurate to 0,001 g). If more than 5 % of the sediment consists of particles                        > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined.                  

###### 2.1.3.3.5.*Extraction and preparation of the flotate*: after recovery of the sediment with the method described above, two phases should remain in the separation funnel: a liquid                           one consisting of tetrachloroethylene and a solid one made of floating material. This solid phase is the flotate and shall                           be recovered by pouring off completely tetrachloroethylene from the funnel by opening the stopcock. By inverting the separation                           funnel, the flotate shall be transferred into a large Petri dish and air dried in a fumehood. If more than 5 % of the flotate                           consists of particles > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined.

###### 2.1.3.3.6.*Preparation of raw material*: a portion of at least 5 g of the ground sub-sample shall be prepared. If more than 5 % of the material consists of particles                           > 0,50 mm, it shall be sieved at 0,25 mm and the two resulting fractions shall be examined.

###### 2.1.3.4.Preparation of samples consisting of fat or oil

The following protocol shall be followed for the preparation of samples consisting of fat or oil:                  
          -  if the fat is solid, it shall be warmed in a oven until it is liquid.                         
          -  by using a pipette, 40 ml of fat or oil shall be transferred from the bottom of the sample to a centrifugation tube.                         
          -  centrifuge during 10 minutes at 4 000 r.p.m.                         
          -  if the fat is solid after centrifugation, it shall be warmed in an oven until it is liquid.                         
          -  repeat the centrifugation during 5 minutes at 4 000 r.p.m.                         
          -  by using a small spoon or a spatula, one half of the decanted impurities shall be transferred to microscopic slides for examination,                              Glycerol is recommended as mounting medium.                         
          -  the remaining impurities shall be used for preparing the sediment as described in point 2.1.3.3.                         

###### 2.1.3.5.Use of staining reagents

In order to facilitate the correct identification of the constituents of animal origin, the operator may use staining reagents                        during the sample preparation in accordance with guidelines issued by the EURL-AP and published on its website.                  

In case Alizarin Red solution is used to colour the sediment, the following protocol shall apply:                  
          -  the dried sediment shall be transferred into a glass test tube and rinsed twice with approximately 5 ml of ethanol (each time                              a vortex of 30 s shall be used, the solvent shall be let settle about 1 min 30 s and poured off).                         
          -  the sediment shall be bleached by adding at least 1 ml sodium hypochlorite solution. The reaction shall be allowed to continue                              for 10 min. The tube shall be filled with water, the sediment shall be let settle 2-3 min, and the water and the suspended                              particles shall be poured off gently.                         
          -  the sediment shall be rinsed twice more with about 10 ml of water (a vortex shall be used for 30 s, let settle, and pour off                              the water each time).                         
          -  2 to 10 drops of the Alizarin Red solution shall be added and the mixture shall be vortexed. The reaction shall be let occur                              for 30 s and the coloured sediment shall be rinsed twice with approximately 5 ml ethanol followed by one rinse with acetone                              (each time a vortex of 30 s shall be used, the solvent shall be let settle about 1 min and poured off).                         
          -  the coloured sediment shall be dried.                         

###### 2.1.4.*Microscopic examination*

###### 2.1.4.1.Slide preparation

Microscopic slides shall be prepared from the sediment and, depending of operator’s choice, from either the flotate or the                        raw material. In case sieving has been used during the sample preparation, the two resulting fractions (the fine and the coarse                        one) shall be prepared. Test portions of fractions spread on slides shall be representative of the whole fraction.                  

A sufficient number of slides shall be prepared in order to ensure that a complete examination protocol as laid down in point                        2.1.4.2 can be carried-out.                  

Microscopic slides shall be mounted with the adequate mounting medium in accordance with the SOP established by the EURL-AP                        and published on its website. The slides shall be covered with coverslips.                  

###### 2.1.4.2.Observation protocols for the detection of animal particles in compound feed and feed material

The prepared microscopic slides shall be observed in accordance with the observation protocols laid down in diagram 1 for                        compound feed and feed materials other than pure fishmeal, or in diagram 2 for pure fishmeal.                  

The microscopic observations shall be conducted using the compound microscope on the sediment and, depending of the operator’s                        choice, either on the flotate or on the raw material. The stereomicroscope may be used in addition to the compound microscope                        for the coarse fractions. Each slide shall be screened entirely at various magnifications.                  

The minimum numbers of slides to be observed at each step of the observation protocol shall be strictly respected unless the                        entire fraction material does not permit to reach the stipulated slide number. No more than 6 slides per determination shall                        be observed.                  

In order to facilitate the identification of the particles’ nature and origin, the operator may use support tools like decision                        support systems, image libraries and reference samples.                  

###### *[X1Diagram 1***Observation protocol for the detection of animal particles in compound feed and feed material other than fishmeal]**

                  

###### *Diagram 2***Observation protocol for the detection of animal particles in fishmeal**

###### 2.1.4.3.Number of determinations

If following a first determination carried out in accordance with the observation protocol laid down in diagram 1 or diagram                        2 as relevant, no animal particle of a given nature (i.e. terrestrial animal or fish) is detected, no additional determination                        is necessary and the result of the analysis shall be reported using the terminology laid down in point 2.1.5.1.                  

If, following a first determination carried out in accordance with the observation protocols laid down in diagram 1 or in                        diagram 2 as relevant, the total number of animal particles of a given nature (i.e. terrestrial animal or fish) detected ranges                        from 1 to 5, a second determination shall be performed from a new 50 g sub-sample. If, following this second determination,                        the number of animal particles of this given nature detected ranges from 0 to 5, the result of the analysis shall be reported                        using the terminology laid down in point 2.1.5.2., else a third determination shall be carried out from a new 50 g sub-sample.                        Nevertheless, if following the first and the second determination, the sum of the particles of a given nature detected over                        the two determinations is higher than 15, no additional determination is necessary and the result of the analysis shall be                        directly reported using the terminology laid down in point 2.1.5.3. If, following the third determination, the sum of the                        animal particles of a given nature detected over the three determinations is higher than 15, the result of the analysis shall                        be reported using the terminology laid down in point 2.1.5.3. Otherwise, the result of the analysis shall be reported using                        the terminology laid down in point 2.1.5.2.                  

If following a first determination carried out in accordance with the observation protocols laid down in diagram 1 or in diagram                        2 as relevant, more than 5 animal particles of a given nature (i.e. terrestrial animal or fish) are detected, the result of                        the analysis shall be reported using the terminology laid down in point 2.1.5.3.                  

###### 2.1.5.*Expression of the results*

When reporting the results, the laboratory shall indicate on which type of material the analysis has been carried-out (sediment,                        flotate or raw material) and how many determinations have been carried-out.                  

The laboratory report shall at least contain information on the presence of constituents derived from terrestrial animals                        and from fish.                  

The different situations shall be reported in the following ways.                  

###### 2.1.5.1.No animal particle of a given nature detected:

          -  as far as was discernible using a light microscope, no particle derived from terrestrial animals was detected in the submitted                              sample,                         
          -  as far as was discernible using a light microscope, no particle derived from fish was detected in the submitted sample.                         

###### 2.1.5.2.Between 1 and 5 animal particles of a given nature detected on average:

          -  as far as was discernible using a light microscope, no more than 5 particles derived from terrestrial animals were detected                              on average per determination in the submitted sample. The particles were identified as ... [bone, cartilage, muscle, hair, horn...].                              This low level presence, being below the limit of detection of the microscopic method, means that a risk of false positive                              result cannot be excluded.                         

Or, as relevant,                  
          -  as far as was discernible using a light microscope, no more than 5 particles derived from fish were detected on average per                              determination in the submitted sample. The particles were identified as ... [fishbone, fish scale, cartilage, muscle, otolith,                              gill...]. This low level presence, being below the limit of detection of the microscopic method, means that a risk of false                              positive result cannot be excluded.                         

In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or                        kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction                        may be the sign of an environmental contamination.                  

###### 2.1.5.3.More than 5 animal particles of a given nature detected on average

          -  as far as was discernible using a light microscope, more than 5 particles derived from terrestrial animals were detected on                              average per determination in the submitted sample. The particles were identified as ... [bone, cartilage, muscle, hair, horn...].                         

Or, as relevant,                  
          -  as far as was discernible using a light microscope, more than 5 particles derived from fish were detected on average per determination                              in the submitted sample. The particles were identified as ... [fishbone, fish scale, cartilage, muscle, otolith, gill...].                         

In case of sample pre-sieving, the laboratory report shall mention in which fraction (sieved fraction, pelleted fraction or                        kernels) the animal particles have been detected insofar as the detection of animal particles only in the sieved fraction                        may be the sign of an environmental contamination.                  

##### 2.2.**PCR**

###### 2.2.1.*Principle*

Deoxyribonucleic acid (DNA) fragments of animal origin which may be present in feed materials and compound feed are detected                        by a genetic amplification technique through PCR, targeting species-specific DNA sequences.                  

The PCR method first requires a DNA extraction step. The amplification step shall be applied afterwards to the so-obtained                        DNA extract, in order to detect the animal species targeted by the assay.                  

###### 2.2.2.*Reagents and equipment*

###### 2.2.2.1.Reagents

###### 2.2.2.1.1.Reagents for DNA extraction step

Only reagents approved by the EURL-AP and published on its website shall be used.                  

###### 2.2.2.1.2.Reagents for genetic amplification step

###### 2.2.2.1.2.1.Primers and probes

Only primers and probes with sequences of oligonucleotides validated by the EURL-AP shall be used(6).                  

###### 2.2.2.1.2.2.Master Mix

Only Master Mix solutions which do not contain reagents susceptible to lead to false results due to presence of animal DNA                        shall be used(7).                  

###### 2.2.2.1.2.3.Decontamination reagents

###### 2.2.2.1.2.3.1.Hydrochloric acid solution (0,1 N)

###### 2.2.2.1.2.3.2.Bleach (solution of sodium hypochlorite at 0,15 % of active chlorine)

###### 2.2.2.1.2.3.3.Non-corrosive reagents for decontaminating costly devices like analytical balances (e.g. DNA EraseTM of MP Biomedicals)

###### 2.2.2.2.Equipment

###### 2.2.2.2.1.Analytical balance with an accuracy of 0,001 g

###### 2.2.2.2.2.Grinding equipment

###### 2.2.2.2.3.Thermocycler enabling real-time PCR

###### 2.2.2.2.4.Microcentrifuge for microfuge tubes

###### 2.2.2.2.5.Set of micropipettes allowing to pipet from 1 μl up to 1 000 μl

###### 2.2.2.2.6.Standard molecular biology plastic-ware: microfuge tubes, filtered plastic tips for micropipettes, plates suitable for the                           thermocycler.

###### 2.2.2.2.7.Freezers to store samples and reagents

###### 2.2.3.*Sampling and sample preparation*

###### 2.2.3.1.Sampling

A representative sample, taken in accordance with the provisions laid down in Annex I, shall be used.                  

###### 2.2.3.2.Sample preparation

The preparation of laboratory samples up to DNA extraction shall comply with the requirements set out in Annex II. At least                        50 g of the sample shall be sub-sampled for analysis and subsequently ground.                  

The sample preparation shall be performed in a room different from the ones dedicated to DNA extraction and to genetic amplification                        reactions as described by ISO 24276.                  

Two test portions of at least 100 mg each shall be prepared.                  

###### 2.2.4.*DNA extraction*

The DNA extraction shall be performed on each test portion prepared using the SOP established by the EURL-AP and published                        on its website.                  

Two extraction controls shall be prepared for each extraction series as described by ISO 24276.                  
          -  an extraction blank control,                         
          -  a positive DNA extraction control.                         

###### 2.2.5.*Genetic amplification*

The genetic amplification shall be performed using the methods validated for each species requiring identification. These                        methods are laid down in the SOP established by the EURL-AP and published on its website. Each DNA extract shall be analysed                        at least at two different dilutions in order to evaluate inhibition.                  

Two amplification controls shall be prepared per species target as described by ISO 24276.                  
          -  a positive DNA target control shall be used for each plate or series of PCR assays,                         
          -  an amplification reagent control (also called no template control) shall be used for each plate or series of PCR assays.                         

###### 2.2.6.*Interpretation and expression of results*

When reporting the results, the laboratory shall indicate at least the weight of the test portions used, the extraction technique                        used, the number of determinations carried-out and the limit of detection of the method.                  

Results shall not be interpreted and reported if the positive DNA extraction control and the positive DNA target controls                        do not provide positive results for the target under assay while the amplification reagent control is negative.                  

In case results from the two test portions are not consistent, at least the genetic amplification step shall be repeated.                        If the laboratory suspects that the DNA extracts can be the cause of the inconsistency, a new DNA extraction and a subsequent                        genetic amplification shall be performed before interpreting the results.                  

The final expression of the results shall be based on the integration and the interpretation of the results of the two test                        portions in accordance with the SOP established by the EURL-AP and published on its website.                  

###### 2.2.6.1.Negative result

A negative result shall be reported as follows:                  

No DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted                        by the assay).                  

###### 2.2.6.2.Positive result

A positive result shall be reported as follows:                  

DNA from X was detected in the submitted sample (with X being the animal species or group of animal species that is targeted                        by the assay).’                  

Editorial Information

X1Substituted by Corrigendum to Commission Regulation (EU) No 51/2013 of 16 January 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 (Official Journal                                 of the European Union L 20 of 23 January 2013).

1

OJ L 165, 30.4.2004, p. 1.                           

2

OJ L 147, 31.5.2001, p. 1.                           

3

OJ L 300, 14.11.2009, p. 1.                           

4

OJ L 54, 26.2.2009, p. 1.                           

5

http://eurl.craw.eu/

6

The list of these primers and probes for each animal species targeted by the assay is available on the EURL-AP website.

7

Examples of Master Mixes that are functional are available on the EURL-AP website.