Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests.

Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1-H4, H6-H8, H10-H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.