2008; Spackman & Suarez, 2008). It has therefore served as the target region for these
subtypes. Spackman et al. (2002) demonstrated specific detection of these subtypes, but
cautioned that their H5 and H7 primer/probe sequences had been designed for the detection of
North American H5 and H7 isolates and might not be suitable for all H5 and H7 isolates. This
proved to be the case. Slomka et al. (Slomka et al., 2007) described modification of the H5
oligonucleotide sequences used by Spackman et al. (2002) to enable the detection of the Asian
lineage H5N1 subtype and other Eurasian H5 subtypes that have been isolated within the past
decade in both poultry and wild birds. Validated real-time RT-PCR protocols for the
simultaneous detection and typing of H5, H7 and H9 RNA have been developed (Monne et al.,
2008). These validated Eurasian real-time RT-PCR have proven valuable in the investigation of
many H5N1 HPAI clinical specimens and other subtypes submitted to International Reference
Laboratories from Europe, Africa and Asia since autumn 2005 (Monne et al., 2008; Slomka et
al., 2007). Each set of primers and probes needs to be validated against a diverse set of viruses
to make the test applicable to in diverse avian species, and in viruses from broad geographic
areas and time periods.