title: A rapid, single-step multiplex reverse transcription-PCR assay for the detection of human H1N1, H3N2, and B influenza viruses.
abstract: BACKGROUND
  Influenza is a viral respiratory pathogen responsible for frequent seasonal epidemics. There are currently three major human influenza viruses in global circulation, H1N1, H3N2 and B.
  
  
  OBJECTIVE
  A one-step multiplex reverse transcription (RT)-polymerase chain reaction (PCR) assay targeting the HA1 segment of the human hemagglutinin gene was developed as a rapid surveillance method.
  
  
  METHODS
  A researcher-blind study was performed using 112 randomly selected, culture-positive clinical samples collected through the Department of Defense (Global Emerging Infectious Surveillance (DOD-GEIS) influenza network during the 2000-2001 influenza season. Three subtype specific primer sets capable of producing PCR products with base-pair lengths of 585, 402 and 290 corresponding to influenza H1, H3, and B subtypes, respectively, were utilized together in a one step, one tube, reaction.
  
  
  RESULTS
  Multiplex primers were able to simultaneously type, and subtype 100% (112/112) of positive cultures.
  
  
  CONCLUSIONS
  The results confirm that this assay is a highly sensitive and timely surveillance tool for rapid detection and simultaneous subtyping of clinical influenza specimens isolated worldwide.
authors.affiliation: Molecular Epidemiology Branch, 2601 West Gate Road, Brooks AFB, San Antonio, TX 78235-5241, USA. luke.daum@brooks.af.mil
authors.forename: Luke T
authors.initials: LT
authors.lastname: Daum
authors.forename: Linda C
authors.initials: LC
authors.lastname: Canas
authors.forename: Cassandra A
authors.initials: CA
authors.lastname: Schadler
authors.forename: Veronica A H
authors.initials: VA
authors.lastname: Ujimori
authors.forename: William B
authors.initials: WB
authors.lastname: Huff
authors.forename: William J
authors.initials: WJ
authors.lastname: Barnes
authors.forename: Kenton L
authors.initials: KL
authors.lastname: Lohman
mesh_terms: D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics*; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype; D053122:Influenza A Virus, H3N2 Subtype; D009980:Influenza A virus / Q000235:genetics / Q000302:isolation & purification*; D009981:Influenza B virus / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus