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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RT-PCR on HA cleavage site
Sequencing of the PCR product
Alignment with other haemaglutinin sequences using bio informatic software or a blast search on genebank
HA1-HA2 cleavage site on avian isolates
Prototype motif forcleavage by ubiquitous
Serial dilutions of allantoic fluid
AIV and NDV free chicken red blood cells 1%
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)