title: Development of multiplex rt-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens.
abstract: We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.
authors.affiliation: College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea.
authors.forename: Hee Kyoung
authors.initials: HK
authors.lastname: Chang
authors.forename: Jeung Hyun
authors.initials: JH
authors.lastname: Park
authors.forename: Min-Suk
authors.initials: MS
authors.lastname: Song
authors.forename: Taek-Kyu
authors.initials: TK
authors.lastname: Oh
authors.forename: Seok-Young
authors.initials: SY
authors.lastname: Kim
authors.forename: Chul-Jung
authors.initials: CJ
authors.lastname: Kim
authors.forename: Hyunggee
authors.initials: H
authors.lastname: Kim
authors.forename: Moon-Hee
authors.initials: MH
authors.lastname: Sung
authors.forename: Heon-Seok
authors.initials: HS
authors.lastname: Han
authors.forename: Youn-Soo
authors.initials: YS
authors.lastname: Hahn
authors.forename: Young-Ki
authors.initials: YK
authors.lastname: Choi
mesh_terms: D000818:Animals; D001717:Birds / Q000821:virology; D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000821:virology; D007723:Korea; D009439:Neuraminidase / Q000235:genetics*; D009976:Orthomyxoviridae Infections / Q000662:veterinary / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000821:virology
chemical_list: D017931:DNA Primers; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; D009439:Neuraminidase