title: Multiplex assay for subtyping avian influenza A viruses by cDNA hybridization and adapter-mediated amplification.
abstract: Multiple subtypes of influenza A viruses circulating in animals must be closely monitored to understand their risk to humans and animal populations. Many molecular-based subtyping methods require constant monitoring of viral genomes for primer and/or probe mismatches and are prone to primer-primer interactions. This report presents a new approach that involves target enrichment through cDNA hybridization followed by adapter-mediated amplification for subtyping influenza virus (AmASIV). As a proof of concept, the AmASIV assay was multiplexed to specifically detect and differentiate influenza A virus subtypes (H5, N5, N7, and N9) in a single reaction without cross-recognition of nontarget subtypes or influenza B virus. The limit of detection (LOD) of AmASIV, as measured by 50 % egg-infective dose per reaction (EID50/reaction), was comparable to that of singleplex TaqMan® qPCR assays with LODs of 10(-0.6) (H5), 10(2) (N5), 10(-0.3) (N7), and 10(-0.5) (N9) EID50/reaction. The AmASIV will strengthen animal influenza virus surveillance and laboratory capacity to improve prevention and control of influenza.
authors.affiliation: Influenza Division, Virology, Surveillance and Diagnosis Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, MS-D30, Atlanta, GA, 30333, USA.
authors.forename: Genyan
authors.identifier: http://orcid.org/0000-0003-4631-6302
authors.initials: G
authors.lastname: Yang
authors.affiliation: Influenza Division, Virology, Surveillance and Diagnosis Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, MS-D30, Atlanta, GA, 30333, USA.
authors.forename: Joyce
authors.initials: J
authors.lastname: Jones
authors.affiliation: Influenza Division, Virology, Surveillance and Diagnosis Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, MS-D30, Atlanta, GA, 30333, USA.
authors.forename: Yunho
authors.initials: Y
authors.lastname: Jang
authors.affiliation: Influenza Division, Virology, Surveillance and Diagnosis Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, MS-D30, Atlanta, GA, 30333, USA. eou8@cdc.gov.
authors.forename: C Todd
authors.initials: CT
authors.lastname: Davis
mesh_terms: D000818:Animals; D001717:Birds; D060005:Genotyping Techniques / Q000379:methods*; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics*; D005585:Influenza in Birds / Q000821:virology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012680:Sensitivity and Specificity
keywords: AmASIV; Influenza; Subtyping; Surveillance