{
  "url": "https://openlegislation.uk/eu-directive-2009-156/annex-IV/part-B/division-1/division-2/division-2.1",
  "json_url": "https://openlegislation.uk/eu-directive-2009-156/annex-IV/part-B/division-1/division-2/division-2.1.json",
  "legislation": "eudr/2009/156",
  "legislation_title": "Council Directive 2009/156/EC of 30 November 2009 on animal health conditions governing the movement and importation from third countries of equidae (codified version) (Text with EEA relevance)",
  "legislation_url": "https://openlegislation.uk/eu-directive-2009-156",
  "path": "annex/IV/part/B/division/1/division/2/division/2.1",
  "kind": "division",
  "number": "2.1",
  "heading": "Group-specific real-time RT-PCR by Agüero et al., 2008",
  "text": "This group-specific real-time RT-PCR targets VP7 of the AHSV and is able to detect all known AHSV serotypes and strains currently circulating. It has been employed with very good results by the participating national reference laboratories of the European Union Member States in the proficiency tests annually organised by the European Union Reference Laboratory for the period 2009-2015. Moreover, in an international ring trial organised in 2015 in the framework of the OIE reference laboratories network this protocol was ranked very high amongst others.\nPrimer and probe sequences for the detection of AHSV species viruses:\n— forward Primer 5′-CCA-GTA-GGC-CAG-ATC-AAC-AG-3′\n— reverse Primer 5′-CTA-ATG-AAA-GCG-GTG-ACC-GT-3′\n— MGB-TaqMan probe 5′-FAM-GCT-AGC-AGC-CTA-CCA-CTA-MGB-3′\n2.1.1. Primer stock concentration is diluted to a working concentration of 8 μM ( ‘ primer working stock 8 μM ’ ) whereas probe is diluted to a working concentration of 50 μM ( ‘ probe working stock 50 μM ’ ). A test plate layout should be designed and loaded into the real time PCR machine software. Using the layout as a guide, 2,5 μl of each primer working stock 8 μM is added to each well that will contain RNA samples, positive and/or negative controls (final concentration of the primer will be 1 μM in the 20 μl RT-PCR mix). The plate is held on ice.\n2.1.2. 2 μl of isolated RNA (test samples and positive control), or 2 μl of RNAse-free water in negative reaction controls, is mixed with forward and reverse primers. This mixture is denatured by heating at 95 °C for 5 minutes, followed by rapid cooling on ice for at least 5 minutes.\n2.1.3. An appropriate volume of real time one-step RT-PCR master mix for the number of samples to be tested is prepared following manufacturer's instructions. 0,1 μl of probe working stock 50 μM is added to each well containing RNA samples (final concentration of the probe will be 0,25 μM in each well containing RNA samples). 13 μl of real time one-step RT-PCR master mix is distributed in each well on the PCR plate containing the denatured primers and RNA.\n2.1.4. The plate is placed in a real time thermal cycler programmed for reverse transcription and cDNA amplification/fluorescence detection. Amplification conditions consist of a first reverse-transcription step at 48 °C for 25 minutes, followed by 10 minutes at 95 °C ( ‘ hot start ’ ) and 40 cycles of 15 seconds at 95 °C, 35 seconds at 55 °C and 30 seconds at 72 °C (or 40 cycles at 97 °C for 2 seconds and 55 °C for 30 seconds if reagents and thermocycler allowing fast reactions are used). Fluorescence data are acquired at the end of the 55 °C step.\n2.1.5. The assay is considered not valid if atypical amplification curves are obtained, and must be repeated.\nSamples are considered positives, if the Ct value (cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold) is lower than or equal to the defined Ct threshold (35) within 40 PCR cycles (Ct ≤ 35).\nSamples are considered inconclusive, if the Ct value is higher than the defined Ct threshold (35) within 40 PCR cycles (Ct > 35).\nSamples are considered negative, if a horizontal amplification curve is obtained which does not cross the threshold line within 40 PCR cycles.",
  "extent": "E+W+S+N.I.",
  "status": null,
  "version": "current",
  "annotations": [
    {
      "type": "F",
      "label": "F1",
      "text": "Substituted by Commission Implementing Decision (EU) 2016/1840 of 14 October 2016 amending Annex IV to Council Directive 2009/156/EC as regards methods for African horse sickness diagnosis (notified under document C(2016) 6509) (Text with EEA relevance)."
    },
    {
      "type": "footnote",
      "label": "f1",
      "text": "Agüero M., Gomez-Tejedor C., Angeles Cubillo M., Rubio C., Romero E. and Jimenez-Clavero A. (2008). Real-time fluorogenic reverse transcription polymerase chain reaction assay for detection of African horse sickness virus. J. Vet. Diagn. Invest., 20, 325-328."
    }
  ],
  "outstanding_effects": [],
  "provenance": {
    "source": "legislation.gov.uk",
    "source_url": "https://www.legislation.gov.uk/eudr/2009/156/annex/IV/part/B/division/1/division/2/division/2.1",
    "retrieved_at": "2026-11-01T15:22:04.142412+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."
}