{
  "url": "https://openlegislation.uk/eu-directive-2006-56/annex-I/appendix-6",
  "json_url": "https://openlegislation.uk/eu-directive-2006-56/annex-I/appendix-6.json",
  "legislation": "eudr/2006/56",
  "legislation_title": "Commission Directive 2006/56/EC of 12 June 2006 amending the Annexes to Council Directive 93/85/EEC on the control of potato ring rot",
  "legislation_url": "https://openlegislation.uk/eu-directive-2006-56",
  "path": "annex/I/appendix/6",
  "kind": "appendix",
  "number": "6",
  "heading": "Validated PCR protocol and reagents",
  "text": "Note:\nPreliminary testing should permit reproducible detection of at least 103 to 104 cells of C. m. sepedonicus per ml of sample extract.\nPreliminary testing should also show no false positive results with a panel of selected bacterial strains.\n1.\nMultiplex PCR protocol with internal PCR control (Pastrik, 2000)\n1.1.\nOligonucleotide primers\nForward primer PSA-1\n5′- ctc ctt gtg ggg tgg gaa aa -3′\nReverse primer PSA-R\n5′- tac tga gat gtt tca ctt ccc c -3′\nForward primer NS-7-F\n5′- gag gca ata aca ggt ctg tga tgc -3′\nReverse Primer NS-8-R\n5’- tcc gca ggt tca cct acg ga -3’\nExpected amplicon size from C. m. subsp. sepedonicus template DNA = 502 bp (PSA-primer set).\nExpected amplicon size from the 18S rRNA internal PCR control = 377 bp (NS-primer set).\n1.2.\nPCR reaction mix\nMethods were validated using Taq polymerase from Perkin Elmer (AmpliTaq or Gold) and Gibco BRL.\nConcentration of primers NS-7 F and NS-8-R were optimised for potato heel end core extraction using the homogenisation method and DNA purification according to Pastrik (2000) (see section 6.1.a) and 6.2). Re-optimisation of reagent concentrations will be required if extraction by shaking or other DNA isolation methods are used.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n15,725 µl\n10x PCR buffer (15 mM MgCl2)\n2,5 µl\n1x (1,5 mM MgCl2)\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\nd-nTP mix (20 mM)\n0,125 µl\n0,1 mM\nPrimer PSA-1 (10 µM)\n0,5 µl\n0,2 µM\nPrimer PSA-R (10 µM)\n0,5 µl\n0,2 µM\nPrimer NS-7-F (10 µM)\n0,1 µl\n0,04 µM\nPrimer NS-8-R (10 µM)\n0,1 µl\n0,04 µM\nTaq polymerase (5 U/µl)\n0,2 µl\n1,0 U\nSample volume\n5,0 µl\nTotal volume\n25,0 µl\n1.3.\nPCR reaction conditions\nRun the following programme:\n1 cycle of:\n(i)\n3 minutes at 95 °C (denaturation of template DNA)\n10 cycles of:\n(ii)\n1 minute at 95 °C (denaturation of template DNA)\n(iii)\n1 minute at 64 °C (annealing of primers)\n(iv)\n1 minute at 72 °C (extension of copy)\n25 cycles of:\n(v)\n30 seconds at 95 °C (denaturation of template DNA)\n(vi)\n30 seconds at 62 °C (annealing of primers)\n(vii)\n1 minute at 72 °C (extension of copy)\n1 cycle of:\n(viii)\n5 minutes at 72 °C (final extension)\n(ix)\nhold at 4 °C.\nNote:\nThis programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) (iv), (v), (vi) and (vii) may be required for use with other models.\n1.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from C. m. subsp. sepedonicus DNA produce a distinctive restriction fragment length polymorphism with enzyme Bgl II after incubation at 37 °C for 30 minutes. The restriction fragments obtained from C. m. subsp. sepedonicus-specific fragment are 282 bp and 220 bp in size.\n2.\nPreparation of the Loading buffer\n2.1.\nBromphenol blue (10 %-stock solution)\nBromphenol blue\n5 g\nDistilled Water (bidest)\n50 ml\n2.2.\nLoading buffer\nGlycerol (86 %)\n3,5 ml\nBromphenol blue (5.1)\n300 µl\nDistilled Water (bidest)\n6,2 ml\n3.\n10x Tris Acetate EDTA (TAE) buffer, pH 8,0\nTris buffer\n48,4 g\nGlacial acetic acid\n11,42 ml\nEDTA (disodium salt)\n3,72 g\nDistilled water\n1,00 L\nDilute to 1x before use.\nAlso commercially available (e.g. Invitrogen or equivalent).",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "footnote",
      "label": "f1",
      "text": "Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq or Gold) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f2",
      "text": "Concentration of primers NS-7 F and NS-8-R were optimised for potato heel end core extraction using the homogenisation method and DNA purification according to Pastrik (2000) (see section 6.1.a) and 6.2). Re-optimisation of reagent concentrations will be required if extraction by shaking or other DNA isolation methods are used."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/2006/56/annex/I/appendix/6",
    "retrieved_at": "2026-11-01T15:22:35.204179+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."
}