Dataset Information

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.

SUBMITTER: Kawai Y

PROVIDER: S-EPMC3266250 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

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

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

PloS one 20120125 1

<h4>Background</h4>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.<h4>Methodology</h4>To address the clinical need for rapid diagnosis, we have developed a new method, the "RT-SmartAmp assay", to r ...[more]

PMID: 22295077

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Project description:BACKGROUND:Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube. METHODS:Clinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay. RESULTS:The limitation of detection (LOD) of the multiplex assay was 5.4 × 10-2 50% tissue culture infective dose (TCID50) per reaction or 4.8 × 101 copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10-2 TCID50 per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10-2 TCID50 per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples. CONCLUSIONS:These findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.

Dataset Information

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

Background

Methodology

Results and conclusions

Publications

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

Similar Datasets

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