Molecular Detection and Identification of Influenza Viruses by Oligonucleotide Microarray Hybridization

ABSTRACT

Microarrays of virus-specific oligonucleotides may provide a method of screening samples for the presence or absence of a large variety of viruses simultaneously. Influenza viruses are ideal for evaluating such microarrays because of their genetic and host diversity, and the availability of an extensive sequence database. A collection of 476 influenza virus-specific oligonucleotides was spotted onto glass slides as probes. Viral RNAs were reverse transcribed and amplified by PCR, and the products were labeled with cyanine dyes. The presence of viruses and their identities were determined by hybridization. The fluorescence intensities of oligonucleotide spots were highly reproducible within each slide and satisfactorily proportional between experiments. However, the intensities of probe spots completely complementary to target sequences varied from background to saturation. The variations did not correlate with base composition, nucleotide sequence, or internal secondary structures. Therefore, thresholds for determining whether hybridization to a spot should be judged as positive were assigned individually. Considering only positive spots from probes predicted to be monospecific for influenza virus species, subtype, host source, or gene segment, this method made correct identifications at the species, hemagglutinin subtype, and gene segment levels. Monospecific neuraminidase (NA) subtype probes were insufficiently diverse to allow confident NA subtype assignment. Incorporating positive spots from polyspecific probes into the identification scheme gave similar results. Overall, the results demonstrate the potential of microarray-based oligonucleotide hybridization for multiple virus detection.