avian influenza diagnostic further analysis
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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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Presentation Transcript
laboratory analyses procedure
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

hemagglutinine and hpai
Hemagglutinine and HPAI

Probe

H5PRO

H5-Kha-1

H5-Kha-3

H5LH1

H5RH1

HA-875F

HA-1114R

J3

B2a

Site

de clivage

HA Gene

sequencing
Sequencing

H5Kha

Sequencing of the PCR product

Alignment with other haemaglutinin sequences using bio informatic software or a blast search on genebank

slide5

Virulent Strains

HA2

HA1

Cleavage site

HA1-HA2 cleavage site on avian isolates

slide6

Cleavage only withtrypsin-like enzyme

Prototype motif forcleavage by ubiquitous

furane

isolation in embryonated chicken eggs
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%

isolation in embryonated chicken eggs1
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
detection of h5 or h7 subtypes
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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