title: Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex).
abstract: This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻⁵(= 280ELD₅₀) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻⁴(=2800 ELD₅₀). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.
authors.affiliation: Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen 518010, China. bioit@163.com
authors.forename: Zhi-feng
authors.initials: ZF
authors.lastname: Qin
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.forename: Ti-kang
authors.initials: TK
authors.lastname: Lu
authors.forename: Shao-ling
authors.initials: SL
authors.lastname: Zeng
authors.forename: Qun-yi
authors.initials: QY
authors.lastname: Hua
authors.forename: Qing-yan
authors.initials: QY
authors.lastname: Ling
authors.forename: Shu-kun
authors.initials: SK
authors.lastname: Chen
authors.forename: Jian-qiang
authors.initials: JQ
authors.lastname: Lv
authors.forename: Cai-hong
authors.initials: CH
authors.lastname: Zhang
authors.forename: Bing
authors.initials: B
authors.lastname: Cheng
authors.forename: Zhou-xi
authors.initials: ZX
authors.lastname: Ruan
authors.forename: Ying-zuo
authors.initials: YZ
authors.lastname: Bi
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Giambrone
authors.forename: Hong-zhuan
authors.initials: HZ
authors.lastname: Wu
mesh_terms: D000818:Animals; D001717:Birds; D017931:DNA Primers / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D009980:Influenza A virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis / Q000821:virology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods
chemical_list: D017931:DNA Primers; D012367:RNA, Viral