Confirmation of the H5N1 subtype was achieved using a
standard RT-PCR. Genomic RNAs were isolated from allantoic fluid,
after digestion with proteinase K in 0.1% SDS, using the RNeasy
Mini Kit (Qiagen). RT-PCR was carried out using the SuperScriptTM
III One-Step RT-PCR System with Platinum1 Taq DNA Polymerase
(Invitrogen). Reactions included 0.2 mM dNTPs, 1.6 mM MgSO4 in
the provided buffer, and 600mM of each of the standard primers,
Matrix, HA-H5 and NA-N1, described previously (WHO, 2005).