title: Detection of avian influenza viruses and differentiation of H5, H7, N1, and N2 subtypes using a multiplex microsphere assay.
abstract: In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase-PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 x 10(5) to 1 x 10(9) copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 x 10(2.5) to 1 x 10(3.2) 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.
authors.affiliation: Poultry Diagnostic and Research Center, Department of Population Health, College of Veterinary Medicine, 953 College Station Road, Athens, GA 30602, USA.
authors.forename: Teneema
authors.initials: T
authors.lastname: Kuriakose
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Hilt
authors.forename: Mark W
authors.initials: MW
authors.lastname: Jackwood
mesh_terms: D000818:Animals; D002645:Chickens; D002988:Cloaca / Q000821:virology; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis* / Q000821:virology; D008863:Microspheres; D009960:Oropharynx / Q000821:virology; D011201:Poultry Diseases / Q000175:diagnosis* / Q000821:virology; D012367:RNA, Viral / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000662:veterinary; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral