title: Singleplex real-time RT-PCR for detection of influenza A virus and simultaneous differentiation of A/H1N1v and evaluation of the RealStar influenza kit.
abstract: BACKGROUND
  A novel influenza A virus, subtype A/H1N1v emerged in April 2009 and caused the first influenza pandemic of the 21st century. Reliable detection and differentiation from seasonal influenza viruses is mandatory for appropriate case management as well as public health.
  
  
  OBJECTIVE
  To develop and technically validate a novel one-step real-time RT-PCR assay which can be used for influenza A virus screening and subtyping of A/H1N1v in a singleplex fashion. To assess the clinical performance of a novel commercial influenza RT-PCR kit based on the in-house version.
  
  
  METHODS
  A real-time RT-PCR assay targeting the matrix gene of influenza A viruses was developed and validated using in vitro transcribed RNA derived from influenza A/H1N1v, A/H1N1 and A/H3N2 virus as well as plaque-quantified influenza A/H1N1v, A/H1N1 and A/H3N2 virus samples. After validation of the in-house version the commercial RealStar kit was used to assess the clinical performance and specificity on a panel of influenza viruses including A/H1N1v, A/H1N1, swine A/H1N1, A/H3N2, avian A/H5N1 as well as patient specimens.
  
  
  RESULTS
  The lower limit of detection of the in-house version was 2149, 1376 and 2994 RNA copies/ml for A/H1N1v, A/H1N1 and A/H3N2, respectively. The RealStar kit displayed 100% sensitivity and specificity and could reliably discriminate influenza A viruses from A/H1N1v. No cross reaction with swine A/H1N1 and A/H1N2 was observed with the RealStar A/H1N1v specific probe.
  
  
  CONCLUSIONS
  Both assays demonstrated high sensitivity and specificity and might assist in the diagnosis of suspected influenza cases.
authors.affiliation: Department of Virology, University of Freiburg, Hermann-Herder Str. 11, 79104 Freiburg, Germany. marcus.panning@uniklinik-freiburg.de
authors.forename: Marcus
authors.initials: M
authors.lastname: Panning
authors.forename: Sigrid
authors.initials: S
authors.lastname: Baumgarte
authors.forename: Thomas
authors.initials: T
authors.lastname: Laue
authors.forename: Sibylle
authors.initials: S
authors.lastname: Bierbaum
authors.forename: Sabine
authors.initials: S
authors.lastname: Raith
authors.forename: Jan Felix
authors.initials: JF
authors.lastname: Drexler
authors.forename: Angelika
authors.initials: A
authors.lastname: Helmer
authors.forename: Valeria
authors.initials: V
authors.lastname: Falcone-Kapper
authors.forename: Georg
authors.initials: G
authors.lastname: Kochs
authors.forename: Hartmut
authors.initials: H
authors.lastname: Campe
authors.forename: Daniela
authors.initials: D
authors.lastname: Huzly
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Eis-Hübinger
authors.forename: Christian
authors.initials: C
authors.lastname: Drosten
mesh_terms: D001483:Base Sequence; D003429:Cross Reactions; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics* / Q000302:isolation & purification*; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D057230:Limit of Detection; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D014763:Viral Matrix Proteins / Q000235:genetics
chemical_list: C488936:M1 protein, Influenza A virus; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D014763:Viral Matrix Proteins