Development of Microsphere-Based Multiplex Branched DNA Assay for Detection and Differentiation of Avian Influenza Virus Strains†

We developed and evaluated a multiplex branched DNA assay for the detection and subtyping of avian influenza (AI) virus strains. The assay successfully detected all 94 AI virus strains of 15 different hemagglutinin (HA) subtypes tested while simultaneously differentiating 24 North American H5, 11 Eurasian H5, and 11 H7 strains. Our study demonstrates for the first time that a branched DNA method can detect targets that show a great amount of sequence variation. Avian influenza (AI) viruses are type A influenza viruses of the Orthomyxoviridae family (15). Influenza virus is typed based on the serological reactions of internal proteins (nucleoprotein and matrix 1) and is further classified into 16 hemagglutinin (HA) and nine neuraminidase (NA) subtypes, respectively. Among the 16 HA subtypes, detection of H5 and H7 subtypes has been emphasized because of their outbreak history and potential of becoming highly pathogenic AI (HPAI) viruses (14). In addition to the economic impact that HPAI virus can cause, the viruses have also been considered to be a publi