title: Typing (A/B) and subtyping (H1/H3/H5) of influenza A viruses by multiplex real-time RT-PCR assays.
abstract: In this study, a specific and sensitive one-step multiplex real-time RT-PCR was developed in two assays by using primers and a number of specific locked nucleic acid (LNA)-mediated TaqMan probes which increase the thermal stability of oligonucleotides. The first assay consisted of primers and probes specific to the matrix (M1) gene of influenza A virus, matrix (M1) gene of influenza B virus and GAPDH gene of host cells for typing of influenza virus and verification by an internal control, respectively. The other assay employed primers and probes specific to the hemagglutinin gene of H1, H3 and H5 subtypes in order to identify the three most prominent subtypes of influenza A capable of infecting humans. The specificity results did not produce any cross reactivity with other respiratory viruses or other subtypes of influenza A viruses (H2, H4 and H6-H15), indicating the high specificity of the primers and probes used. The sensitivity of the assays which depend on the type or subtype being detected was approximately 10 to 10(3)copies/microl that depended on the types or subtypes being detected. Furthermore, the assays demonstrated 100% concordance with 35 specimens infected with influenza A viruses and 34 specimens infected with other respiratory viruses, which were identified by direct nucleotide sequencing. In conclusion, the multiplex real-time RT-PCR assays have proven advantageous in terms of rapidity, specificity and sensitivity for human specimens and thus present a feasible and attractive method for large-scale detection aimed at controlling influenza outbreaks.
authors.affiliation: Center of Excellence in Clinical Virology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand.
authors.forename: Kamol
authors.initials: K
authors.lastname: Suwannakarn
authors.forename: Sunchai
authors.initials: S
authors.lastname: Payungporn
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Chieochansin
authors.forename: Rujipat
authors.initials: R
authors.lastname: Samransamruajkit
authors.forename: Alongkorn
authors.initials: A
authors.lastname: Amonsin
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Songserm
authors.forename: Arunee
authors.initials: A
authors.lastname: Chaisingh
authors.forename: Pornchai
authors.initials: P
authors.lastname: Chamnanpood
authors.forename: Salin
authors.initials: S
authors.lastname: Chutinimitkul
authors.forename: Apiradee
authors.initials: A
authors.lastname: Theamboonlers
authors.forename: Yong
authors.initials: Y
authors.lastname: Poovorawan
mesh_terms: D000818:Animals; D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics; D009976:Orthomyxoviridae Infections / Q000821:virology; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral