Detection and subtyping of influenza A virus based on a short oligonucleotide microarray.
We report the design and characterization of a microarray with 46 short virus-specific oligonucleotides for detecting influenza A virus of 5 subtypes: H1N1, H1N2, H3N2, H5N1, and H9N2. A unique combination of 3 specific modifications was introduced into the microarray assay: (1) short probes of 19 to 27 nucleotides, (2) simple amplification of full-length hemagglutinin and neuraminidase cDNAs with universal primers, and (3) Klenow-mediated labeling and further amplification of the samples before hybridization. The assay correctly and specifically detected and subtyped 11 different influenza A isolates from human, avian, and swine species representing the 5 subtypes. When tested with 225 clinical samples, 20 were detected to be positive using our microarray-based assay, whereas only 10 were positive by the conventional culture method. The entire analysis was completed within 7 h. Thus, these modifications result in a specific, sensitive, and rapid microarray assay and may be used for constructing microarrays for the detection of all influenza subtypes and strains.
Li
Xihan
X
Institute of Virology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu 210093, China.
Qi
Xian
X
Miao
Lv
L
Wang
Yu
Y
Liu
Fangzheng
F
Gu
Hongwei
H
Lu
Sangwei
S
Yang
Yonghua
Y
Liu
Fenyong
F
D005453:Fluorescence; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000235:genetics; D057221:Influenza A Virus, H1N2 Subtype / Q000145:classification / Q000235:genetics; D053122:Influenza A Virus, H3N2 Subtype / Q000145:classification / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification / Q000235:genetics; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics; D007251:Influenza, Human / Q000175:diagnosis / Q000821:virology*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes; D012367:RNA, Viral