Commission Implementing Regulation (EU) 2015/1375Show full title

D. Magnetic stirrer method for pooled sample digestion/'on filter isolation' and larva detection by a latex agglutination test U.K.

This method is only considered equivalent for the testing of meat of domestic swine.

1. Apparatus and reagents U.K.

(a)

Knife or scissors and tweezers for cutting specimens.

(b)

Trays marked off into 50 squares, each of which can hold samples of approximately 2 g of meat, or other tools giving equivalent guarantees as regards the traceability of the samples.

(c)

A blender with a sharp chopping blade. Where the samples are larger than 3 g, a meat mincer with openings of 2 to 4 mm or scissors must be used. In the case of frozen meat or tongue (after removal of the superficial layer, which cannot be digested), a meat mincer is necessary and the sample size will need to be increased considerably.

(d)

Magnetic stirrers with thermostatically controlled heating plate and Teflon-coated stirring rods approximately 5 cm long.

(e)

Glass beakers, capacity 3 litres.

(f)

Sieves, mesh size 180 microns, external diameter 11 cm, with stainless steel mesh.

(g)

Steel filtration apparatus for 20 μm mesh filters with a steel funnel.

(h)

Vacuum pump.

(i)

Metal or plastic tanks, capacity 10 to 15 litres, to collect the digestive juice.

(j)

A 3D gyratory rocker.

(k)

Aluminium foil.

(l)

25 % hydrochloric acid.

(m)

Pepsin, strength: 1:10 000 NF (US National Formulary) corresponding to 1:12 500 BP (British Pharmacopoeia) and to 2 000 FIP (Fédération internationale de pharmacie), or stabilised liquid pepsin with minimum 660 European Pharmacopoeia units/ml.

(n)

Tap water heated to 46 to 48 °C.

(o)

A balance accurate to 0,1 g.

(p)

Pipettes of different sizes (1, 10 and 25 ml), micropipettes according to the latex agglutination manufacturer's instructions and pipette holders.

(q)

20 microns nylon mesh filters of a diameter that fits with the filtration system.

(r)

Plastic or steel forceps of 10 to 15 cm.

(s)

Conical vials of 15 ml.

(t)

A pestle with a Teflon or steel conical tip to fit in the conical vials.

(u)

A thermometer accurate to 0,5 °C within the range 1 to 100 °C.

(v)

Latex agglutination cards of the Trichin-L antigen test kit validated under the code No EURLP_D_001/2011.

(w)

Buffer solution with preservative (sample diluent) of the Trichin-L antigen test kit validated under the code No EURLP_D_001/2011.

(x)

Buffer supplemented with preservative (negative control) of the Trichin-L antigen test kit validated under the code No EURLP_D_001/2011.

(y)

Buffer supplemented with Trichinella spiralis antigens and preservative (positive control) of the Trichin-L antigen test kit validated under the code No EURLP_D_001/2011.

(z)

Buffer with polystyrene particles coated with antibodies supplemented with preservative (latex beads) of the Trichin-L antigen test kit validated under the code No EURLP_D_001/2011.

(aa)

Disposable sticks.

2. Collecting of specimens U.K.

As stipulated in Chapter I(2).

3. Procedure U.K.

I. For complete pools (100 g of samples at a time) U.K.

(a)

16 ± 0,5 ml of 25 % hydrochloric acid (0,2 % final) is added to a 3 litre beaker containing 2,0 litres ± 200 ml of tap water, preheated to 46 to 48 °C; a stirring rod is placed in the beaker, the beaker is placed on the preheated plate and the stirring is started.

(b)

10 ± 1 g of powder pepsin (or 30 ± 3 ml of liquid pepsin) is added.

(c)

100-115 g of samples collected in accordance with point 2 are chopped in the blender, with 150 ± 15 ml of preheated digestion buffer.

(d)

The chopped meat is transferred to the 3 litre beaker containing the water, pepsin and hydrochloric acid.

(e)

The mincing insert of the blender is immersed repeatedly in the digestion fluid in the beaker and the blender bowl is rinsed with a small quantity of digestion fluid to remove any meat still adhering.

(f)

The beaker is covered with aluminium foil.

(g)

The magnetic stirrer must be adjusted so that it maintains a constant temperature of 44 to 46 °C throughout the operation. During stirring, the digestion fluid must rotate at a sufficiently high speed to create a deep whirl without splashing.

(h)

The digestion fluid is stirred until the meat particles disappear (approximately 30 minutes). The stirrer is then switched off and the digestion fluid is poured through the sieve into the sedimentation funnel. Longer digestion times may be necessary (not exceeding 60 minutes) in the processing of certain types of meat (tongue, game meat, etc.).

(i)

The digestion process is considered satisfactory if not more than 5 % of the starting sample weight remains on the sieve.

(j)

The 20 microns nylon mesh filter is placed on the filtration support. The conical filtration steel funnel is fixed to the support with the block system and the steel sieve of 180 microns mesh size is placed on the funnel. The vacuum pump is connected with the filtration support and with the metal or plastic tank, to collect the digestive fluid.

(k)

Stirring is stopped and the digestion fluid is poured into the filtration funnel through the sieve. The beaker is rinsed with approximately 250 ml of warm water. The rinsing liquid is poured into the filtration ramp after the digested fluid has been successfully filtrated.

(l)

The filtration membrane is taken with the forceps, holding it by an edge. The filtration membrane is folded (minimal) in four and put in the 15 ml conical tube. The choice of conical tube must be adapted to the pestle.

(m)

The filtration membrane is pushed at the bottom of the 15 ml conical tube with the help of the pestle and strongly pressed by doing approximately 20 successive back and forth movements with the pestle which should be positioned inside the filtration membrane folding according to the manufacturer's instructions.

(n)

0,5 ± 0,01 ml of sample diluents is added into the 15 ml conical tube by pipette and the filtration membrane is homogenised with the pestle by doing successive low amplitude back and forth movements for approximately 30 seconds, avoiding abrupt movements to limit liquid splashes according to the manufacturer's instructions.

(o)

Each sample, the negative control, and the positive control, are dispensed into different fields of the agglutination card by pipettes, according to the manufacturer's instructions.

(p)

The latex beads are added into each field of the agglutination card by a pipette, according to the manufacturer's instructions, without making them come into contact with the sample/s and controls. In each field, the latex beads are then gently mixed with a disposable stick until the homogeneous liquid covers the entire field.

(q)

The agglutination card is put on the 3D rocker and is rocked for 10 ± 1 minutes according to the manufacturer's instructions.

(r)

After the time established by the manufacturer's instructions, the rocking is stopped and the agglutination card is put on a plane surface and the reaction results are read immediately, according to the manufacturer's instructions. In the case of a positive sample, the beads aggregates must appear. In the case of a negative sample, the suspension remains homogeneous without beads aggregates.

II. Pools of less than 100 g as set out in Chapter I(3)(II) U.K.

For pools of less than 100 g, the procedure set out in Chapter I(3)(II) must be followed.

III. Positive or doubtful results U.K.

Where examination of a collective sample produces a positive or uncertain latex agglutination result, a further 20 g sample is taken from each swine in accordance with Chapter I(2)(a). The 20 g samples from five swine are pooled and examined using the method described in Section I. In this way samples from 20 groups of five swine must be examined.

When a positive latex agglutination is obtained from a group of five swine, further 20 g samples are collected from the individuals in the group and each is examined separately using the method described in Section I.

When a positive or uncertain latex agglutination result is obtained, at least 20 g of swine muscle must be sent to the national reference laboratory for confirmation using one of the methods described in Chapter I.

Parasite samples must be kept in 90 % ethyl alcohol for conservation and identification at species level at the F1 ... national reference laboratory.

Textual Amendments

F1 Words in Annex 1 Ch. 2(D) para. 3(3) omitted (31.12.2020) by virtue of S.I. 2019/640, reg. 51M(b) (as inserted by The Food and Feed Hygiene and Safety (Miscellaneous Amendments etc.) (EU Exit) Regulations 2020 (S.I. 2020/1504), regs. 1(2), 9(14))

After parasite collection, positive fluids must be decontaminated by heating to at least 60 °C.

IV. Cleaning and decontamination procedure after a positive or doubtful result U.K.

When the examination of a collective or individual sample produces a positive or doubtful latex agglutination result, all material in contact with meat (blender bowl and blade, pestle, beaker, stirring rod, temperature sensor, conical filtration funnel, sieve and forceps) must be carefully decontaminated by soaking for few seconds in warm water (65 to 90 °C). Meat residues or inactivated larvae that could remain on their surface may be removed with a clean sponge and tap water. If required, a few drops of detergent can be added for degreasing equipment. It is then recommended to rinse each piece thoroughly to remove all traces of detergent.