[Typing and subtyping avian influenza virus using DNA microarrays].
OBJECTIVE
Outbreaks of highly pathogenic avian influenza (HPAI) virus has caused great economic loss to the poultry industry and resulted in human deaths in Thailand and Vietnam since 2004. Rapid typing and subtyping of viruses, especially HPAI from clinical specimens, are desirable for taking prompt control measures to prevent spreading of the disease. We described a simultaneous approach using microarray to detect and subtype avian influenza virus (AIV).


METHODS
We designed primers of probe genes and used reverse transcriptase PCR to prepare cDNAs of AIV M gene, H5, H7, H9 subtypes haemagglutinin genes and N1, N2 subtypes neuraminidase genes. They were cloned, sequenced, reamplified and spotted to form a glass-bound microarrays. We labeled samples using Cy3-dUTP by RT-PCR, hybridized and scanned the microarrays to typing and subtyping AIV.


RESULTS
The hybridization pattern agreed perfectly with the known grid location of each probe, no cross hybridization could be detected. Examinating of HA subtypes 1 through 15, 30 infected samples and 21 field samples revealed the DNA microarray assay was more sensitive and specific than RT-PCR test and chicken embryo inoculation.


CONCLUSIONS
It can simultaneously detect and differentiate the main epidemic AIV. The results show that DNA microarray technology is a useful diagnostic method.
Yang
Zhongping
Z
Animal Influenza Laboratory of Ministry of Agriculture and National Key Laboratory of Veterinary Biotechnology of Harbin Veterinary Research Institute of CAAS, Harbin 150001, China. yangzhongping19812@126.com
Wang
Xiurong
X
Tian
Lina
L
Wang
Yu
Y
Chen
Hualan
H
D000818:Animals; D015342:DNA Probes / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D015342:DNA Probes