title: One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RT-SmartAmp assay and its clinical validation.
abstract: BACKGROUND
  In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society.
  
  
  METHODS
  To address the clinical need for rapid diagnosis, we have developed a new method, the "RT-SmartAmp assay", to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses.
  
  
  CONCLUSIONS
  We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.
authors.affiliation: Omics Science Center, RIKEN Yokohama Institute, Tsurumi-ku, Yokohama, Japan.
authors.forename: Yuki
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authors.lastname: Kawai
authors.forename: Yasumasa
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authors.lastname: Kimura
authors.forename: Alexander
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authors.forename: Yasushi
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authors.forename: Takayuki
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authors.lastname: Saito
authors.forename: Norio
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authors.forename: Masatsugu
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authors.lastname: Chikahira
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authors.lastname: Hayashi
authors.forename: Sei-ichi
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authors.lastname: Tsuruoka
authors.forename: Tokumichi
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authors.lastname: Toguchi
authors.forename: Yoshitomo
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authors.lastname: Saito
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Ban
authors.forename: Shinyu
authors.initials: S
authors.lastname: Izumi
authors.forename: Hideko
authors.initials: H
authors.lastname: Uryu
authors.forename: Koichiro
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authors.lastname: Kudo
authors.forename: Yuko
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authors.lastname: Sakai-Tagawa
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kawaoka
authors.forename: Aizan
authors.initials: A
authors.lastname: Hirai
authors.forename: Yoshihide
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authors.lastname: Hayashizaki
authors.forename: Toshihisa
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authors.lastname: Ishikawa
mesh_terms: D000368:Aged; D002648:Child; D017931:DNA Primers / Q000235:genetics; D024882:Drug Resistance, Viral; D005260:Female; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity*; D007251:Influenza, Human / Q000453:epidemiology* / Q000821:virology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D053139:Oseltamivir / Q000494:pharmacology; D058873:Pandemics; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013997:Time Factors
chemical_list: D017931:DNA Primers; C543345:H1N1 virus hemagglutinin; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; D053139:Oseltamivir; D012194:RNA-Directed DNA Polymerase