PLoS ONE (Jan 2012)

One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RT-SmartAmp assay and its clinical validation.

  • Yuki Kawai,
  • Yasumasa Kimura,
  • Alexander Lezhava,
  • Hajime Kanamori,
  • Kengo Usui,
  • Takeshi Hanami,
  • Takahiro Soma,
  • Jean-Étienne Morlighem,
  • Satomi Saga,
  • Yuri Ishizu,
  • Shintaro Aoki,
  • Ryuta Endo,
  • Atsuko Oguchi-Katayama,
  • Yasushi Kogo,
  • Yasumasa Mitani,
  • Takefumi Ishidao,
  • Chiharu Kawakami,
  • Hideshi Kurata,
  • Yumiko Furuya,
  • Takayuki Saito,
  • Norio Okazaki,
  • Masatsugu Chikahira,
  • Eiji Hayashi,
  • Sei-ichi Tsuruoka,
  • Tokumichi Toguchi,
  • Yoshitomo Saito,
  • Toshiaki Ban,
  • Shinyu Izumi,
  • Hideko Uryu,
  • Koichiro Kudo,
  • Yuko Sakai-Tagawa,
  • Yoshihiro Kawaoka,
  • Aizan Hirai,
  • Yoshihide Hayashizaki,
  • Toshihisa Ishikawa

DOI
https://doi.org/10.1371/journal.pone.0030236
Journal volume & issue
Vol. 7, no. 1
p. e30236

Abstract

Read online

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. METHODOLOGY: 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. RESULTS AND 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.