Development of a Reverse Transcription-Loop-Mediated Isothermal Amplification Assay for Detection of Pandemic (H1N1) 2009 Virus as a Novel Molecular Method for Diagnosis of Pandemic Influenza in Resource-Limited Settings

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Abstract
This paper reports on the development of a one-step, real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the hemagglutinin (HA) gene for the rapid molecular-based detection of pandemic (H1N1) 2009 virus. The detection limit of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay was same as that of the currently used real-time reverse transcription-PCR method. The assay detected the pandemic (H1N1) 2009 virus HA gene in 136 RNA samples extracted from nasopharyngeal swab specimens from Japanese and Vietnamese patients. No cross-reactive amplification with the RNA of other seasonal influenza viruses was observed, and the detection of specific viral genome targets in clinical specimens was achieved in less than 40 min. The sensitivity and specificity of the pandemic (H1N1) 2009 virus HA-specific RT-LAMP assay obtained in this study were 97.8% and 100%, respectively. Use of the (H1N1) 2009 virus HA-specific RT-LAMP assay will enable the faster and easier diagnosis of pandemic (H1N1) 2009 virus infection, especially in resource-limited situations in developing countries.