[Microarray for detecting and subtyping of circulating influenza A virus].
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.


CONCLUSIONS
The results show that the microarray is a useful diagnostic method with high specificity and sensitivity, and could be used for influenza surveillance.
Li
Xihan
X
Institute of Virology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China. xihanli81@yahoo.com.cn
Wang
Yu
Y
Liu
Fangzheng
F
Gu
Hongwei
H
Yang
Yonghua
Y
D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction