{
  "url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-6",
  "json_url": "https://openlegislation.uk/eu-directive-1998-57/annex-II/appendix-6.json",
  "legislation": "eudr/1998/57",
  "legislation_title": "Council Directive 98/57/EC of 20 July 1998 on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.",
  "legislation_url": "https://openlegislation.uk/eu-directive-1998-57",
  "path": "annex/II/appendix/6",
  "kind": "appendix",
  "number": "6",
  "heading": "Validated PCR protocols and reagents",
  "text": "NB: Preliminary testing should permit reproducible detection of 10 3 to 10 4 cells of R. solanacearum per ml of sample extract.\nPreliminary testing should also show no false positive results with a panel of selected bacterial strains (see Appendix 3).\n1.\nPCR protocol of Seal et al. (1993)\n1.1.\nOligonucleotide primers\nForward primer OLI-1\n5′-GGG GGT AGC TTG CTA CCT GCC-3′\nReverse primer Y-2\n5′-CCC ACT GCT GCC TCC CGT AGG AGT-3′\nExpected amplicon size from R. solanacearum template DNA = 288 bp\n1.2.\nPCR reaction mix\nMethod was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n17,65 µl\n10X PCR buffer (15 mM MgCl 2 )\n2,5 µl\n1X (1,5 mM MgCl 2 )\ndNTP mix (20 mM)\n0,25 µl\n0,2 mM\nPrimer OLI-1 (20 µM)\n1,25 µl\n1µM\nPrimer Y-2 (20 µM)\n1,25 µl\n1µM\nTaq polymerase (5U/µl)\n0,1 µl\n0,5 U\nSample volume\n2,0 µl\nTotal volume\n25 µl\n1.3.\nPCR reaction conditions\nRun the following programme:\n1 cycle of:\n(i)\n2 minutes at 96 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n20 seconds at 94 °C (denaturation of template DNA)\n(iii)\n20 seconds at 68 °C (annealing of primers)\n(iv)\n30 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n10 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme was optimised for use with a Perkin Elmer 9600 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n1.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Ava II after incubation at 37 °C.\n2.\nPCR protocol of Pastrik and Maiss (2000)\n2.1.\nOligonucleotide primers\nForward primer Ps-1\n5′- agt cga acg gca gcg ggg g -3′\nReverse primer Ps-2\n5′- ggg gat ttc aca tcg gtc ttg ca -3′\nExpected amplicon size from R. solanacearum template DNA = 553 bp.\n2.2.\nPCR reaction mix\nMethods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nN.B. Originally optimised for MJ Research PTC 200 thermocycler with Gibco Taq Polymerase.\nPerkin Elmer AmpliTaq and buffer can also be used at the same concentrations.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n16,025 µl\n10X PCR buffer\n2,5 µl\n1X (1,5 mM MgCl 2 )\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\nd-nTP mix (20 mM)\n0,125 µl\n0,1 mM\nPrimer Ps-1 (10 µM)\n0,5 µl\n0,2 µM\nPrimer Ps-2 (10 µM)\n0,5 µl\n0,2 µM\nTaq polymerase (5U/µl)\n0,1 µl\n0,5 U\nSample volume\n5,0 µl\nTotal volume:\n25,0 µl\n2.3.\nPCR reaction conditions\nRun the following programme:\n1 cycle of:\n(i)\n5 minutes at 95 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n30 seconds at 95 °C (denaturation of template DNA)\n(iii)\n30 seconds at 68 °C (annealing of primers)\n(iv)\n45 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n5 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n2.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Taq I after incubation at 65 °C for 30 minutes. The restriction fragments obtained from R. solanacearum -specific fragment are 457 bp and 96 bp in size.\n3.\nMultiplex PCR protocol with internal PCR control (Pastrik et al. , 2002)\n3.1.\nOligonucleotide primers\nForward primer RS-1-F\n5′- ACT AAC GAA GCA GAG ATG CAT TA -3′\nReverse primer RS-1-R\n5′- CCC AGT CAC GGC AGA GAC T -3′\nForward primer NS-5-F\n5′- AAC TTA AAG GAA TTG ACG GAA G -3′\nReverse primer NS-6-R\n5′- GCA TCA CAG ACC TGT TAT TGC CTC -3′\nExpected amplicon size from R. solanacearum template DNA = 718 bp (RS-primer set)\nExpected amplicon size from the 18S rRNA internal PCR control = 310 bp (NS-primer set).\n3.2.\nPCR reaction mix\nMethods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nConcentration of primers NS-5-F and NS-6-R were optimised for potato heel end core extraction using the homogenisation method and DNA purification according to Pastrik (2000) (see Section VI.A.6.1.a.). 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\n12,625 µl\n10X PCR buffer (15 mM MgCl 2 )\n2,5 µl\n1X (1,5 mM MgCl 2 )\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\nd-nTP mix (20 mM)\n0,125 µl\n0,1 mM\nPrimer RS-1-F (10 µM)\n2,0 µl\n0,8 µM\nPrimer RS-1-R (10 µM)\n2,0 µl\n0,8 µM\nPrimer NS-5-F (10 µM)\n0,15 µl\n0,06 µM\nPrimer NS-6-R (10 µM)\n0,15 µl\n0,06 µM\nTaq polymerase (5 U/µl)\n0,2 µl\n1,0 U\nSample volume\n5,0 µl\nTotal volume:\n25,0 µl\n3.3.\nPCR reaction conditions\nRun the following programme:\n1 cycle of:\n(i)\n5 minutes at 95 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n30 seconds at 95 °C (denaturation of template DNA)\n(iii)\n30 seconds at 58 °C (annealing of primers)\n(iv)\n45 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n5 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme is optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n3.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes.\n4.\nR. solanacearum biovar-specific PCR protocol (Pastrik et al. , 2001)\n4.1.\nOligonucleotide primers\nForward primer Rs-1-F\n5′- ACT AAC GAA GCA GAG ATG CAT TA -3′\nReverse primer Rs-1-R\n5′- CCC AGT CAC GGC AGA GAC T -3′\nReverse primer Rs-3-R\n5′- TTC ACG GCA AGA TCG CTC -3′\nExpected amplicon size from R. solanacearum template DNA:\nwith Rs-1-F/Rs-1-R = 718 bp\nwith Rs-1-F/Rs-3-R = 716 bp.\n4.2.\nPCR reaction mix\n(a) Biovar 1/2-specific PCR\nMethods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n12,925 µl\n10X PCR Buffer\n2,5 µl\n1X (1,5 mM MgCl 2 )\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\nd-NTP mix (20mM)\n0,125 µl\n0,1 mM\nPrimer Rs-1-F (10 µM)\n2 µl\n0,8 µM\nPrimer Rs-1-R (10 µM)\n2 µl\n0,8 µM\nTaq polymerase (5U/µl)\n0,2 µl\n1 U\nSample volume\n5,0 µl\nTotal volume\n25,0 µl\n(b) Biovar 3/4/5-specific PCR\nMethods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL.\nReagent\nQuantity per reaction\nFinal concentration\nSterile UPW\n14,925 µl\n10X PCR Buffer\n2,5 µl\n1X (1,5 mM MgCl 2 )\nBSA (fraction V) (10 %)\n0,25 µl\n0,1 %\ndNTP mix (20 mM)\n0,125 µl\n0,1 mM\nPrimer Rs-1-F (10 µM)\n1 µl\n0,4 µM\nPrimer Rs-3-R (10 µM)\n1 µl\n0,4 µM\nTaq polymerase (5 U/µl)\n0,2 µl\n1 U\nSample volume\n5,0 µl\nTotal volume\n25,0 µl\n4.3.\nPCR reaction conditions\nRun the following programme for both biovar 1/2- and biovar 3/4/5-specific reactions:\n1 cycle of:\n(i)\n5 minutes at 95 °C (denaturation of template DNA)\n35 cycles of:\n(ii)\n30 seconds at 95 °C (denaturation of template DNA)\n(iii)\n30 seconds at 58 °C (annealing of primers)\n(iv)\n45 seconds at 72 °C (extension of copy)\n1 cycle of:\n(v)\n5 minutes at 72 °C (final extension)\n(vi)\nhold at 4 °C.\nNB: This programme was optimised for use with an MJ Research PTC 200 thermal cycler. Modification of the duration steps of cycles (ii), (iii) and (iv) may be required for use with other models.\n4.4.\nRestriction enzyme analysis of amplicon.\nPCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-1-R produce a distinctive restriction fragment length polymorphism with enzyme Bsm I or an Isoschizomere (e.g. Mva 1269 I) after incubation at 65 °C for 30 minutes. PCR products amplified from R. solanacearum DNA using primers Rs-1-F and Rs-3-R have no restriction sites.\n5.\nPreparation of the loading buffer\n5.1.\nBromphenol blue (10 %- stock solution )\nBromphenol blue\n5 g\nDistilled water (bidest)\n50 ml\n5.2.\nLoading buffer\nGlycerol (86 %)\n3,5 ml\nBromphenol blue (5,1)\n300 µl\nDistilled Water (bidest)\n6,2 ml\n6.\n10X Tris Acetate EDTA (TAE) buffer, pH 8.0\nTris buffer\n48,40 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": "F",
      "label": "F1",
      "text": "Substituted by Commission Directive 2006/63/CE of 14 July 2006 amending Annexes II to VII to Council Directive 98/57/EC on the control of Ralstonia solanacearum (Smith) Yabuuchi et al.."
    },
    {
      "type": "footnote",
      "label": "f1",
      "text": "Method was validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f2",
      "text": "Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f3",
      "text": "Methods were validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f4",
      "text": "Concentration of primers NS-5-F and NS-6-R were optimised for potato heel end core extraction using the homogenisation method and DNA purification according to Pastrik (2000) (see Section VI.A.6.1.a.). Re-optimisation of reagent concentrations will be required if extraction by shaking or other DNA isolation methods are used."
    },
    {
      "type": "footnote",
      "label": "f5",
      "text": "Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    },
    {
      "type": "footnote",
      "label": "f6",
      "text": "Methods have been validated using Taq polymerase from Perkin Elmer (AmpliTaq) and Gibco BRL."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/1998/57/annex/II/appendix/6",
    "retrieved_at": "2026-11-01T15:23:06.931886+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."
}