Objective: We designed and characterized an oligonucleotide microarray for detecting and subtyping of circulating influenza A virus including subtypes H1N1, H1N2, H3N2, H5N1 and H9N2.
Methods: Based on the sequences of influenza A viruses obtained from the Influenza Virus Resource database of National Center for Biotechnology Information, 46 oligonucleotides probes and one quality control probe were designed to fabricate the microarray. The full-length cDNAs of hemagglutinin and neuraminidase genes were amplified by RT-PCR using universal primers, and the resulting PCR products were labeled and fragmented using Klenow fragment before hybridized with the microarray. A total of 18 different influenza A virus strains representing 5 subtypes and 186 clinical samples were used to validate the specificity and sensitivity of the microarray.
Results: All 18 strains were accurately detected and subtyped by the microarray and no cross hybridization could be detected. The limit of detection for the microarray was approximately 1 x 10(4) gene copies of in vitro transcribed RNA. Of the 186 clinical samples, 8 were successfully subtyped as H1N1 and 4 were subtyped as H3N2.
Conclusion: The results show that the microarray is a useful diagnostic method with high specificity and sensitivity, and could be used for influenza surveillance.