[Oligonucleotide microarray for subtyping avian influenza virus].
OBJECTIVE
Avian influenza viruses are important human and animal respiratory pathogens and rapid diagnosis of novel emerging avian influenza viruses is vital for effective global influenza surveillance. We developed an oligonucleotide microarray-based method for subtyping all avian influenza virus (16 HA and 9 NA subtypes).


METHODS
In total 25 pairs of primers specific for different subtypes and 1 pair of universal primers were carefully designed based on the genomic sequences of influenza A viruses retrieved from GenBank database. Several multiplex RT-PCR methods were then developed, and the target cDNAs of 25 subtype viruses were amplified by RT-PCR or overlapping PCR for evaluating the microarray. Further 52 oligonucleotide probes specific for all 25 subtype viruses were designed according to published gene sequences of avian influenza viruses in amplified target cDNAs domains, and a microarray for subtyping influenza A virus was developed. Then its specificity and sensitivity were validated by using different subtype strains and 2653 samples from 49 different areas.


RESULTS
The results showed that all the subtypes of influenza virus could be identified simultaneously on this microarray with high sensitivity, which could reach to 2.47 pfu/mL virus or 2.5 ng target DNA. Furthermore, there was no cross reaction with other avian respiratory virus.


CONCLUSIONS
An oligonucleotide microarray-based strategy for detection of avian influenza viruses has been developed. Such a diagnostic microarray will be useful in discovering and identifying all subtypes of avian influenza virus.
Xueqing
Han
H
The Institute of Animal and Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, China. hanxueq@yahoo.com.cn
Xiangmei
Lin
L
Yihong
Hou
H
Shaoqiang
Wu
W
Jian
Liu
L
Lin
Mei
M
Guangle
Jia
J
Zexiao
Yang
Y
D000818:Animals; D001717:Birds; D017931:DNA Primers / Q000235:genetics; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology*; D007251:Influenza, Human / Q000821:virology*; D009439:Neuraminidase / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase