Using short oligonucleotide viral-specific arrays

Microarrays of virus-specific oligonucleotides may provide a method of screening samples simultaneously for the presence or absence of a large variety of viruses. The identification of the virus associated with SARS as a coronavirus occurred in part through the use of a microarray of long oligonucleotides (70-mers) designed to detect human respiratory viruses. Arrays of short oligonucleotides (20-mers), in theory, have the potential of much greater specificity.

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 produced by the OU Health Sciences Center Laboratory for Genomics and Bioinformatics was spotted on glass slides at the OSU Microarray Core Facility by a team of researchers (Srikumar Sengupta, Kenji Onodera, Alexander Lai and Ulrich Melcher) in Dr. Melcher's lab at OSU.

Viral RNAs were reverse transcribed, amplified by the polymerase chain reaction and the products labeled with cyanine dyes. The presence of viruses and their identities were determined by hybridization to the oligonucleotide arrays. The fluorescent intensities of the spots were highly reproducible within each slide and 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, correct identifications at the species, HA subtype, and gene segment levels were made. Incorporating positive spots from polyspecific probes into the identification scheme gave similar results while monospecific NA subtype probes were insufficient to allow confident NA subtype assignment.

Overall, the results demonstrate the potential of microarray-based short oligonucleotide hybridization for multiple virus detection. A manuscript describing these experiments was recently published in the Journal of Clinical Microbiology and can be accessed via the following link:

http://jcm.asm.org/cgi/reprint/41/10/4542