(a)

Sampling for the purposes of Article 3(2)(b) shall consist of:

When selecting those animals, all parts of the consignment must be proportionally represented in the sample. If weak, gaping or freshly dead (not decomposed) animals are present, primarily such animals shall be selected.

(b)

Sampling for the purposes of Article 5(2) shall consist of at least 150 individuals of Crassostrea gigas per sampling points. All farms or mollusc farming areas in the Member State or compartment covered by the programme shall be sampled.

Sampling for the purposes of Article 5(1)(b) shall consist of at least 150 individuals of Crassostrea gigas oysters per compartment.

When selecting those animals, the following criteria shall be taken into account:

(c)

The sampling provided for in Article 5(2) shall be carried out in the period of the year when prevalence of OsHV-1 μvar in the Member State or compartment is known to be maximal. When such data is not available, sampling shall be carried out just after the period when the water temperature exceeds 16 °C or at the time of the year when the temperature normally reaches its yearly maximum.

(d)

The sampling provided for in Article 5(1)(b) shall preferably be carried out in the period of the year described in point c. If samples are collected outside that period of the year, the sampled oysters must be maintained under conditions equivalent to those described in point c for a period suitable for the detection of OsHV-1 μvar, before being tested.

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A closed hood equipped with an UV producing system to eliminate potential contaminations when preparing PCR mix.

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Two complete sets of pipettes (2 μl; 20 μl; 200 μl and 1 000 μl), the first one for DNA extraction, and the second one for PCR mix preparation.

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Three different pipettes: one pipette (2 μl) to dispense samples in PCR mix, one pipette (20 μl) for EB sampling and another pipette (20 μl) to load PCR products in agarose gels.

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Filter pipette tips (2 μl; 20 μl; 200 μl and 1 000 μl) for DNA extraction, PCR mix preparation and sample dispensing.

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Pipette tips (20 μl) to collect EB and to load amplification products in agarose gel.

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A thermal cycler to perform amplifications.

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A horizontal electrophoresis system for PCR products electrophoresis.

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An UV table to observe PCR products after agarose gel electrophoresis.

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A system to acquire pictures of the gels.

(a)

For larvae, pools of 50 mg of the whole animals (including the shell) completed with 200 μl of distilled water are crushed and centrifuged at 1 000 g for 1 minute.

(b)

For spat smaller than or of 6 mm, pools of 300 mg of the whole animals (including the shell) completed with 1 200 μl of distilled water are crushed and centrifuged at 1 000 g for 1 minute.

(c)

For spat between 6 and 15 mm in size, all the soft tissues of each animal are crushed individually.

(d)

For animals bigger than 15 mm, pieces of gills and mantle are isolated.

1.

Place 100 μl of supernatant for samples referred to in (a) and (b) or 10 to 50 mg of tissues for samples referred to point (c) and (d) in a 1,5 ml microcentrifuge tube and add 180 μl of Buffer ATL.

2.

Add 20 μl Proteinase K, mix by vortexing and incubate at 56 °C until the tissue is completely lysed (overnight). Vortex occasionally during incubation to disperse sample. Briefly centrifuge the 1,5 ml microcentrifuge tube to remove drops from the lid.

3.

Add 200 μl Buffer AL to the sample, mix by pulse-vortexing for 15 s and incubate at 70 °C for 10 minutes. Briefly centrifuge the 1,5 ml microcentrifuge tube to remove drops from the lid.

4.

Add 200 μl ethanol (96-100 %) to the sample, and mix by pulse-vortexing for 15 s. Briefly centrifuge the 1,5 ml microcentrifuge tube to remove drops from the lid.

5.

Carefully apply the mixture from step 4 to the QIAamp Spin Column (in a 2 ml collection tube) without wetting the rim. Close the cap and centrifuge at 10 000 rpm for 1 min. Place the QIAamp Spin Column in a clean 2 ml collection tube (provided in the kit) and discard the tube containing the filtrate.

6.

Carefully open the QIAamp Spin Column and add 500 μl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 10 000 rpm for 1 min. Place the QIAamp Spin Column in a clean 2 ml collection tube (provided in the kit) and discard the collection tube containing the filtrate.

7.

Carefully open the QIAamp Spin Column and add 500 μl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (14 000 rpm) for 3 min.

8.

(Optional) Place the QIAamp Spin Column in a new 2ml collection tube (not provided in the kit) and discard the collection tube containing the filtrate. Centrifuge at full speed (14 000 rpm) for 1 min.

9.

Place the QIAamp Spin Column in a clean 1,5 ml microcentrifuge tube (not provided in the kit) and discard the collection tube containing the filtrate. Carefully open the QIAamp Spin Column and add 100 μl of distilled water. Incubate 5 minutes at room temperature and centrifuge at 10 000 rpm for 1 min.

10.

Control the quality and efficacy of the extraction (for example by measuring OD (260 nm) under spectrophotometer or after electrophoresis in agarose gel).

11.

Prepare dilution of your samples in order to have a final DNA concentration of 50-100 ng/μl.

12.

DNA solutions are kept at 4 °C until PCR analyses are performed.

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10 X Buffer (furnished with the Taq DNA polymerase)

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MgCl2 (furnished with the DNA polymerase) (25 mM)

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Taq DNA Polymerase (Goldstar, Eurogentec) 5 U/μl

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dNTP (dATP, dCTP, dGTP, dTTT) Master Mix (20mM) must be diluted 10 fold (at 2 mM) before use

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d H2O (distilled H2O free of DNA and RNA)

CF (10 μM)

CR (10 μM)

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49 μl of this PCR mix is dispensed in each PCR tube

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1 μl of extracted DNA (50-100 ng/μl) is added to each tube

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Negative controls consist of dH2O (1 μl for 49 μl of PCR mix). They aim at detecting potential reactive contamination or working environment. One negative control should be included every 10 samples or after each batch of samples.

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Positive controls consist of plasmidic DNA containing the OsHV-1 target genome region CF-CR. They aim at checking the efficacy of the PCR reaction. One positive control should be included for each PCR analysis. Positive controls are available from the Community Reference Laboratory.

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Initial denaturation: 2 min at 94 °C

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Amplification: 35 cycles (1 min at 94 °C, 1 min at 50 °C and 1 min at 72 °C)

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Final elongation: 5 min at 72 °C

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50 X TAE (can be bought directly ready for use):

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Agarose gel 2,5 % in 1 X TAE

Ethidium bromide (0,5 μg/ml) added after cooling the gel.

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Loading blue dye:

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Molecular weight marker:

SmartLadder SF (Eurogentec): a ready-to-use molecular weight marker including 9 regularly spaced bands from 100 to 1 000 bp.

1.

Weight 2,5 g of agarose, add 100 ml of 1 X TAE and heat until the mix is melted.

2.

After cooling the solution, ethidium bromide is added (5 μl for 100 ml of agarose gel) and the solution is disposed in a specific mould equipped with combs (to form slots).

3.

When gel is polymerised, combs are removed and gel is placed in a horizontal electrophoresis system containing enough 1 X TAE to the cover agarose gel.

4.

10 μl of PCR products are mixed with 2 μl of blue dye (6 X) and disposed in the slots.

5.

One whole is dedicated to the molecular weight marker (5 μl).

6.

A voltage of 50 to 150 volts is applied during 30 min to 1 hour depending on the gel size and thickness.

7.

Gel is observed under UV.

(a)

the number, rate and distribution of molluscs on the farm or mollusc farming area infected;

(b)

distance and density of neighbouring farms or mollusc farming areas;

(c)

proximity to processing establishments, contact farms or contact mollusc farming areas;

(d)

species present at the farms or mollusc farming areas;

(e)

farming practices applied in the affected and the neighbouring farms or mollusc farming areas; and

(f)

hydrodynamic conditions and other factors of epizootiological significance identified.