title: [Oligonucleotide microarray for subtyping avian influenza virus].
abstract: 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.
authors.affiliation: The Institute of Animal and Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100029, China. hanxueq@yahoo.com.cn
authors.forename: Han
authors.initials: H
authors.lastname: Xueqing
authors.forename: Lin
authors.initials: L
authors.lastname: Xiangmei
authors.forename: Hou
authors.initials: H
authors.lastname: Yihong
authors.forename: Wu
authors.initials: W
authors.lastname: Shaoqiang
authors.forename: Liu
authors.initials: L
authors.lastname: Jian
authors.forename: Mei
authors.initials: M
authors.lastname: Lin
authors.forename: Jia
authors.initials: J
authors.lastname: Guangle
authors.forename: Yang
authors.initials: Y
authors.lastname: Zexiao
mesh_terms: 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
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase