Development of PNA-based chromatography for rapid detection of respiratory viruses

• Project/Area Number: 24390146
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Laboratory medicine
• Research Institution: Kyoto Prefectural University of Medicine
• Principal Investigator: NAKAYA TAKAAKI 京都府立医科大学, 医学(系)研究科(研究院), 教授 (80271633)
• Co-Investigator(Kenkyū-buntansha): KAIHATSU Kunihiro 大阪大学, 産業科学研究所, 特任准教授 (70419464)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥18,720,000 (Direct Cost: ¥14,400,000、Indirect Cost: ¥4,320,000); Fiscal Year 2014: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2013: ¥5,330,000 (Direct Cost: ¥4,100,000、Indirect Cost: ¥1,230,000); Fiscal Year 2012: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
• Keywords: 迅速診断 / デバイス / インフルエンザウイルス / ペプチド核酸 / ウイルス検出 / マイクロ流体システム / ウイルス / クロマトデバイス / オンチップ / 感染症

Outline of Final Research Achievements

In this study, we developed a novel chromatography kit for the subtype-specific detection of influenza A viruses. Azobenzene-tethered hairpin-type peptide nucleic acid (Tail-clamp PNA-AZO) possessing a subtype-specific complementary sequence against human influenza A virus, H1-, H3-, and avian H7-subtype, was synthesized and attached to a chromatography strip. The sensitivity of the chromatography was approximately 1.0×104 ffu/kit for H1N1 and H3N2, and 5.0×105 ffu/kit for H7N7, respectively. The PNA-chromatography specifically detected each influenza A virus tested, although it slightly reacted with other subtypes.
Even though further improvement is necessary to use it clinically as a rapid diagnostic kit, this system could be applied for the sequence-specific detection of influenza viruses, including drug-resistant viruses as well as highly-pathogenic avian influenza viruses.

Research Products

• [Journal Article] Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients. (2015)
  • Author(s): 1.Watanabe Y, Arai Y, Daidoji T, Kawashita N, Ibrahim MS, El-Gendy E, Hiramatsu H, Kubota-Kokestu R, Takagi T, Murata T, Takahashi K, Okuno Y, Nakaya T, Suzuki Y, Ikuta K.
  • Journal Title: mBio Volume: 6 Issue: 2
  • DOI: 10.1128/mbio.00081-15
  • Peer Reviewed / Open Access
• [Journal Article] Emergence of infectious malignant thrombocytopenia in Japanese macaques (Macaca fuscata) by SRV-4 after transmission to a novel host. (2015)
  • Author(s): Okamoto M, Miyazawa T, Morikawa S, Ono F, Nakamura S, Sato E, Yoshida T, Yoshikawa R, Sakai K, Mizutani T, Nagata N, Takano J, Okabayashi S, Hamano M, Fujimoto K, Nakaya T, Iida T, Horri T, Miyabe-Nishiwaki T, Watanabe A, Kaneko A, Saito A, Matsui A, Hayakawa T, Suzuki J, Akari H, Matsuzawa T, Hirai H.
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 8850-8850
  • DOI: 10.1038/srep08850
  • Peer Reviewed / Open Access
• [Journal Article] Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH. (2015)
  • Author(s): 3.Daidoji T, Watanabe Y, Ibrahim MS, Yasugi M, Maruyama H, Masuda T, Arai F, Ohba T, Honda A, Ikuta K, Nakaya T
  • Journal Title: J Biol Chem Volume: 290 Issue: 17 Pages: 10627-42
  • DOI: 10.1074/jbc.m114.611327
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Viral detection by high-throughput sequencing (2015)
  • Author(s): 4.Motooka D, Nakamura S, Hagiwara K, Nakaya T
  • Journal Title: Methods Mol Biol Volume: 1236 Pages: 125-34
  • DOI: 10.1007/978-1-4939-1743-3_11
  • ISBN: 9781493917426, 9781493917433
• [Journal Article] A novel immunochromatographic system for easy-to-use detection of group 1 avian influenza viruses with acquired human-type receptor binding specificity (2014)
  • Author(s): Watanabe Y, Ito T, Ibrahim MS, Arai Y, Hotta K, Phuong HVM, Hang NLK Mai LQ, Soda K, Yamaoka M, Poetranto ED, Wulandari L, Hiramatsu H, Daidoji T, Kubota-Koketsu R, Sriwilaijaroen N, Nakaya T, Okuno Y, Takahashi T, Suzuki T, Ito T, Hotta H, Yamashiro T, Hayashi T, Morita K, Ikuta K, Suzuki Y
  • Journal Title: Biosens Bioelectron Volume: 65 Pages: 211-219
  • DOI: 10.1016/j.bios.2014.10.036
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Virus purification and enrichment by hydroxyapatite chromatography on a chip. (2014)
  • Author(s): 6.Niimi M, Masuda T, Kaihatsu K, Kato N, Nakamura S, Nakaya T, Arai F.
  • Journal Title: Sensors and Actuators B: Chemical Volume: 201 Pages: 185-90
  • DOI: 10.1016/j.snb.2014.04.011
  • Peer Reviewed
• [Journal Article] Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses (2014)
  • Author(s): Pan Y, Sasaki T, Kubota-Koketsu R, Inoue Y, Yasugi M, Yamashita A, Ramadhany R, Arai Y, Du A, Boonsathorn N, Ibrahim MS, Daidoji T, Nakaya T, Ono K, Okuno Y, Ikuta K, Watanabe Y
  • Journal Title: Biochem Biophys Res Commun Volume: 450 Issue: 1 Pages: 42-48
  • DOI: 10.1016/j.bbrc.2014.05.060
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Sequence-specific and visual identification of influenza virus gene by azobenzene-tethered bis-peptide nucleic acid (2013)
  • Author(s): K. Kaihatsu, S. Sawada, S. Nakamura, T. Nakaya, T. Yasunaga, N. Kato
  • Journal Title: PLoS ONE Volume: 8 Issue: 5 Pages: e64017-e64017
  • DOI: 10.1371/journal.pone.0064017
  • Peer Reviewed
• [Journal Article] Human monoclonal antibodies broadly neutralizing against influenza B virus. (2013)
  • Author(s): Yasugi, M., Kubota-Koketsu, R., Yamashita, A., Kawashita, N., Du, A., Sasaki, T., Nishimura, M., Misaki, R., Kuhara, M., Boonsathorn, N., Fujiyama, K., Okuno, Y., Nakaya, T., and Ikuta, K.
  • Journal Title: PLoS Pathog. Volume: 9 Issue: 2 Pages: e1003150-e1003150
  • DOI: 10.1371/journal.ppat.1003150
  • Peer Reviewed
• [Journal Article] Emergence of HA mutants during influenza virus pneumonia. (2012)
  • Author(s): Manriquez ME, Nakaya T, Shinya K, et al.
  • Journal Title: Int. J. Clin. Exp. Pathol. Volume: 5(8) Pages: 787-795
  • Peer Reviewed
• [Journal Article] Antigenic analysis of highly pathogenic avian influenza virus H5N1 sublineage co-circulating in Egypt. (2012)
  • Author(s): Watanabe, Y., Ibrahim, M.S., Ellakany, H.F., Kawashita, N., Daidoji, T., Takagi, T., Yasunaga, T., Nakaya, T., and Ikuta, K.
  • Journal Title: J. Gen. Virol. Volume: 93 Issue: 10 Pages: 2215-2226
  • DOI: 10.1099/vir.0.044032-0
  • Peer Reviewed
• [Presentation] 環境中微生物の網羅的探索（メタゲノム解析） (2015)
  • Author(s): 中屋 隆明
  • Organizer: 第2回大気エアロゾルシンポジウム
  • Place of Presentation: 北海道江別市
  • Year and Date: 2015-02-20
  • Invited
• [Presentation] しのび寄る新興感染症における最新の話題 (2014)
  • Author(s): 中屋 隆明
  • Organizer: 第20回京都地域医療フォーラム
  • Place of Presentation: 京都市
  • Year and Date: 2014-09-20
  • Invited
• [Presentation] 鳥インフルエンザ (2012)
  • Author(s): 中屋 隆明
  • Organizer: 日本神経感染症学会
  • Place of Presentation: 京都府京都市
  • Year and Date: 2012-10-20
• [Presentation] インフルエンザウイルスの宿主域拡大戦略 (2012)
  • Author(s): 中屋 隆明
  • Organizer: CREST公開シンポジウム「光が拓く細胞解析の新展開」
  • Place of Presentation: 東京都品川区
  • Year and Date: 2012-08-18
• [Presentation] 新興・再興ウイルス感染症のダイナミクス (2012)
  • Author(s): 中屋 隆明
  • Organizer: びわ湖国際医療フォーラム
  • Place of Presentation: 滋賀県大津市
  • Year and Date: 2012-07-14
• [Presentation] H1N1 2009-HAゲノムのディープシークエンス解析 (2012)
  • Author(s): 中屋 隆明
  • Organizer: インフルエンザ研究者交流の会シンポジウム
  • Place of Presentation: 福島県耶麻郡
  • Year and Date: 2012-05-25
• [Book] 次世代シーケンサー活用術 (2015)
  • Author(s): 中村昇太、飯田哲也・中屋隆明・堀井俊宏
  • Total Pages: 199
  • Publisher: 化学同人