{
  "url": "https://openlegislation.uk/eu-decision-2015-1554/annex-II/part-5/division-I/division-I.3/division-I.3.2",
  "json_url": "https://openlegislation.uk/eu-decision-2015-1554/annex-II/part-5/division-I/division-I.3/division-I.3.2.json",
  "legislation": "eudn/2015/1554",
  "legislation_title": "Commission Implementing Decision (EU) 2015/1554 of 11 September 2015 laying down rules for the application of Directive 2006/88/EC as regards requirements for surveillance and diagnostic methods (notified under document C(2015) 6188) (Text with EEA relevance)",
  "legislation_url": "https://openlegislation.uk/eu-decision-2015-1554",
  "path": "annex/II/part/5/division/I/division/I.3/division/I.3.2",
  "kind": "division",
  "number": "I.3.2",
  "heading": "Polymerase chain reaction (PCR)",
  "text": "Several PCR protocols have been developed and published.\nTwo PCR protocols targeting the small subunit (SSU) rDNA may be used:\n(a)the first one is a conventional PCR that amplify several members of the Haplosporidia including Bonamia spp. Primers designated Bo and Boas are 5′-CAT-TTA-ATT-GGT-CGG-GCC-GC-3′ and 5′-CTG-ATC-GTC-TTC-GAT-CCC-CC-3′ respectively, and amplify a 300 bp product. PCR mixtures contain buffer (500 mM KCl, 100 mM Tris/HCl [pH 9,0 at 25 °C] and 1 % Triton® X-100), 2,5 mM MgCl2, 0,2 mM dNTP mix, 1 μM forward and reverse primers, 0,02 units μl– 1 Taq DNA polymerase, and 0,2 ng μl– 1 of the DNA template in a total volume of 50 μl. Samples shall be denatured in a thermocycler for five minutes at 94 °C before being submitted to 30 cycles (94 °C for one minute, 55 °C for one minute, 72 °C for one minute) followed by a final extension of 10 minutes at 72 °C.\nPositive controls shall consist of genomic DNA from a highly infected host or plasmidic DNA including the target region.\nNegative controls shall consist of genomic DNA from non-infected hosts and PCR reactives without target DNA.\nA positive result shall be positive PCR amplification at the expected size (namely, 300 bp), with all negative controls being negative and all positive controls being positive;\n(b)the second PCR protocol is a SYBR® Green Real time PCR assay. It allows the specific detection of B. ostreae (described below) and may be combined with a SYBR® Green Real time PCR assay allowing the specific detection of B. exitiosa (Ramilo et al. 2013).\nPrimers BOSTRE-F (5′- TTACGTCCCTGCCCTTTGTA-3′) and BOSTRE-R (5′- TCGCGGTTGAATTTTATCGT-3′) amplify a 208 bp product. PCR mixtures contain SYBR® Green Master Mix (1X), 0,3 μM forward and reverse primers, and 200 ng of extracted DNA. Samples shall be denatured in a Real Time Detection System for 10 minutes at 95 °C before being submitted to 35 cycles (95 °C for 30 seconds, 55 °C for 45 seconds and 72 °C for one minute). Melting temperature curve analysis shall be carried out with temperature increments of 0,5 °C/s starting at 55 °C and ending at 95 °C and recording fluorescence at each change of temperature.\nPositive controls shall consist in genomic DNA from a highly infected host or plasmidic DNA including the target region.\nNegative controls shall consist in genomic DNA from non-infected hosts and PCR reactives without target DNA.\nA positive result shall be positive PCR amplification with a unique melting temperature peak (78,25 ± 0,25 °C in the conditions published by Ramilo et al. 2013), with all negative controls being negative and all positive controls being positive.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudn/2015/1554/annex/II/part/5/division/I/division/I.3/division/I.3.2",
    "retrieved_at": "2026-11-01T15:14:14.330569+00:00"
  },
  "licence": "Open Government Licence v3.0",
  "licence_url": "https://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/",
  "attribution": "Contains public sector information licensed under the Open Government Licence v3.0. Source: legislation.gov.uk, The National Archives."
}