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. 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.

DOI: 10.1371/journal.pone.0030236

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@inproceedings{Kawai2012OneStepDO, title={One-Step Detection of the 2009 Pandemic Influenza A(H1N1) Virus by the RT-SmartAmp Assay and Its Clinical Validation}, author={Yuki Kawai and Yasumasa Kimura and Alexander Lezhava and Hajime Kanamori and Kengo Usui and Takeshi Hanami and Takahiro Soma and Jean-{\'E}tienne R L Morlighem and Satomi Saga and Yuri Ishizu and Shintaro Aoki and Ryuta Endo and Atsuko Oguchi-Katayama and Yasushi Kogo and Yasumasa Mitani and Takefumi Ishidao and Chiharu Kawakami and Hideshi Kurata and Yumiko Furuya and Takayuki Saito and Norio Okazaki and Masatsugu Chikahira and Eiji Hayashi and Sei-ichi Tsuruoka and Tokumichi Toguchi and Yoshitomo Saito and Toshiaki Ban and Shinyu Izumi and Hideko Uryu and Koichiro Kudo and Yuko Sakai-Tagawa and Yoshihiro Kawaoka and Aizan Hirai and Yoshihide Hayashizaki and Toshihisa Ishikawa}, booktitle={PloS one}, year={2012} }