Rapid Detection of Novel Influenza A Virus and Seasonal Influenza A (H1N1, H3N2) Viruses by Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP)

• Published in: Kansenshogaku Zasshi Vol. 84; no. 4; pp. 431 - 436
• Main Authors: OHARA, Sachiko, SIMAZU, YUKIE, FUKUDA, Shinji, SHIGEMOTO, Naoki, TANIZAWA, Yukie, TAKAO, Shinichi, KUWAYAMA, Masaru
• Format: Journal Article
• Language: Japanese
• Published: Japan The Japanese Association for Infectious Diseases 01.07.2010
• Subjects: 2009 influenza（H1N1）; Animals; Humans; Influenza A virus - isolation & purification; Influenza A Virus, H1N1 Subtype - isolation & purification; Influenza A Virus, H3N2 Subtype - isolation & purification; influenza A（H1N1）; influenza A（H3N2）; Nucleic Acid Amplification Techniques; Reverse Transcription; reverse transcription-loop-mediated isothermal amplification（RT-LAMP）
• ISSN: 0387-5911; 1884-569X
• DOI: 10.11150/kansenshogakuzasshi.84.431

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay we developed detects novel influenza A (H1N1) of swine origin and seasonal influenza A (H1N1 and H3N2) viruses. Individual primer sets targeting the HA gene for novel H1N1, H1N1, and H3N2 were newly designed to specifically detect these subtypes. No cross-reactions occurred among novel H1N1, H1N1, and H3N2, and 7 respiratory viruses-influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and rhinovirus―had no reaction to 3 RT-LAMP assays. RT-LAMP is assayed at 63℃for 40 min. In our RT-LAMP assay, Eriochrome Black T was added to the reaction mixture as an amplification indicator to detect virus genomes without using real-time turbidimetry. Positive reactions were indicated in blue and negative reactions remained purple. Of 139 samples from suspected novel H1N1 subjects tested by both RT-LAMP and real-time RT-PCR assay, 110 were positive in both assays. Two samples with low copy numbers were positive only in real-time RT-PCR assay. Of 27 novel negative H1N1 samples, 4 were positive for H3N2 on viral isolation and conventional RT-PCR assay. RT-LAMP assay for detecting H3N2 obtained the same findings. Our RT-LAMP assay is thus potentially useful in rapidly detecting influenza A virus such as novel H1N1, H1N1, and H3N2.