Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay

For more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. In this report, we describe a rapid and simple gold nanoparticle (NP)-based genomic microarray assay for specific identification of avian influenza virus H5N1 and its discrimination from other major influenza A virus strains (H1N1, H3N2). Capture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix (M) gene of H1N1, H3N2 and H5N1 viruses, and sequences specific for the hemaglutinin (HA) and neuraminidase (NA) genes of the H5N1 virus. Viral RNA was detected within 2.5 hours using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining in the absence of RNA fragmentation, target amplification, and enzymatic reactions. The lower limit of detection (LOD) of the assay was less than 100 fM for purified PCR fragments and 103 TCID50 units for H5N1 viral RNA. The NP-based microarray assay was able to detect and distinguish H5N1 sequences from those of major influenza A viruses (H1N1, H3N2). The new method described here may be useful for simultaneous detection and subtyping of major influenza A viruses.