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Avian influenza diagnostic further analysis

Avian influenza diagnostic further analysis. Laboratory analyses procedure. Mix. RNA extraction. Sample sorting. Discard Swabs. Influenza A Q RT-PCR : M gene Primers M+25, M-124 and the probe M+25 (Spackman et al, 2002) Brilliant QRT-PCR one-step Master mix kit (Stratagene).

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Avian influenza diagnostic further analysis

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  1. Avian influenza diagnostic further analysis

  2. Laboratory analyses procedure Mix RNA extraction Sample sorting Discard Swabs • Influenza A Q RT-PCR : M gene • Primers M+25, M-124 and the probe M+25 (Spackman et al, 2002) • Brilliant QRT-PCR one-step Master mix kit (Stratagene) Positive samples Positive samples • Influenza H5 and H7 Q RT-PCR : • primers and probes recommended by the reference laboratory VLA (Weybridge, UK) • one-step RT-PCR kit (Qiagen) RT-PCR on HA cleavage site

  3. Hemagglutinine and HPAI Probe H5PRO H5-Kha-1 H5-Kha-3 H5LH1 H5RH1 HA-875F HA-1114R J3 B2a Site de clivage HA Gene

  4. Sequencing H5Kha Sequencing of the PCR product Alignment with other haemaglutinin sequences using bio informatic software or a blast search on genebank

  5. Virulent Strains HA2 HA1 Cleavage site HA1-HA2 cleavage site on avian isolates

  6. Cleavage only withtrypsin-like enzyme Prototype motif forcleavage by ubiquitous furane

  7. Isolation in embryonated chicken eggs • Inoculation of sample in allantoic cavity of SPF embryo chicken eggs of 9 to 11 days • 5 eggs per sample • About 200 µl of sample • Confirmation of isolation after 2 to 7 days of incubation at 37°C • Mortality • Detection of haemagglutinating activity of allantoic fluid: 1/4096 1/2 Serial dilutions of allantoic fluid HA+ HA- 1h + AIV and NDV free chicken red blood cells 1%

  8. Isolation in embryonated chicken eggs • Confirmation of the presence of Influenza A virus by Agar gel immunodiffusion test • Determination of the subtype by inhibition haemagglutination assay with specific antiserums

  9. Detection of H5 or H7 subtypes High variation of haemagglutinin gene Necessary to verify the sequences of the circulating strains to design new primers so that false negative can be avoided ex: design of H5 real-time RT-PCR primers by Spackman et al. (2002) Observation of differences on HA gene between North American Strain and Eurasian strains Modification of H5 primers used in Europe and Africa (Slomka et al. 2007)

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