Development of a highly sensitive RNA virus detection method by artificial nucleoproteins

• Project/Area Number: 15H05229
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 海外学術
• Research Field: Medical genome science
• Research Institution: Osaka University
• Principal Investigator: KAIHATSU KUNIHIRO 大阪大学, 産業科学研究所, 特任准教授(常勤) (70419464)
• Co-Investigator(Kenkyū-buntansha): 黒須 剛 国立感染症研究所, ウイルス第一部, 主任研究官 (70432432)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥15,730,000 (Direct Cost: ¥12,100,000、Indirect Cost: ¥3,630,000); Fiscal Year 2017: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2016: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2015: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
• Keywords: ウイルス / バイオテクノロジー / 遺伝子 / 核酸 / 蛋白質 / デングウイルス / 遺伝子診断 / ペプチド核酸

Outline of Final Research Achievements

At Ministry of Health of Thailand, we analyzed blood sample of dengue infected patients and succeeded to identify (1) the viral serotypes (1-4 type) on the virus infected cultured cells using fluorescent labeled antibodies. Then, (2) EDTA was added to the blood supernatant, and genomic RNA was identified after the viral RNA isolation, double stranded DNA amplification by PCR. (3)dengue virus specific IgM and IgG were detected by anti-IgM and anti-IgG on a plate and detected by ELISA method.
Based on the above described method, viral gene and the protein complex was labeled by chemically-modified antibody. The complex was captured by peptide nucleic acid on the test line in the sequence specific manner. It allow us to identify the presence of dengue virus in the specimen.

Academic Significance and Societal Importance of the Research Achievements

ヒトは、蚊が媒介するデングウイルスに複数回感染すると、デングショック熱やデング出血熱など重篤な感染症状を発症する。従来の抗体診断キットを用いた場合、デングウイルスに複数回感染した検体中の抗原検出感度が低下することがわかった。一方で、本プロジェクトで開発したペプチド核酸を用いれば同ウイルスの遺伝子/蛋白質複合体をその場で検出できることが明らかとなった。これらの知見はデング熱感染重症化の早期処置に有用となる新しい診断手法になりうると期待する。

Research Products

• [Int'l Joint Research] タイ保健省/マヒドン大学(タイ)
• [Int'l Joint Research] タイ保健省/マヒドン大学(タイ)
• [Int'l Joint Research] 保健省医科学局/マヒドン大学(タイ)
• [Journal Article] Design of Tail-Clamp Peptide Nucleic Acid Tethered with Azobenzene Linker for Sequence-Specific Detection of Homopurine DNA (2017)
  • Author(s): Sawada Shinjiro、Takao Toshifumi、Kato Nobuo、Kaihatsu Kunihiro
  • Journal Title: Molecules Volume: 22 Issue: 11 Pages: 1840-1840
  • DOI: 10.3390/molecules22111840
  • Peer Reviewed / Open Access
• [Journal Article] Future Perspective of Nucleic Acid-based Detection of Dengue Virus and its Serotypes (2016)
  • Author(s): K Kaihatsu、E Harada、H Matsumura、 A Takenaka、 N Wichukchinda、 A Sa-Ngarsang、 N Kato
  • Journal Title: Journal of Antivirals & Antiretrovirals Volume: 8 Issue: 2 Pages: 69-72
  • DOI: 10.4172/jaa.1000e132
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Sequence-specific detection and visualization of dengue viral RNA-protein complex by peptide nucleic acid (2017)
  • Author(s): K. Kaihatsu, Emi Harada, A. Takenaka, H. Matsumura, N. Kato
  • Organizer: The 43rd International Symposium on Nucleic Acids Chemistry
  • Int'l Joint Research
• [Presentation] Sequence specific detection of RNA viral gene by chemically-modified peptide nucleic acid (2016)
  • Author(s): 開發 邦宏
  • Organizer: The 6th Euro Virology Congress and Expo
  • Place of Presentation: Madrid, Spain. (Oral)
  • Year and Date: 2016-03-10
• [Presentation] Rapid identification of RNA viruses by peptide nucleic acid chromatography (2015)
  • Author(s): 開發 邦宏
  • Organizer: Pacifichem-2015
  • Place of Presentation: Hawaii, USA. (Poser)
  • Year and Date: 2015-12-14
• [Book] Detecting pathogenic viruses (2018)
  • Author(s): Kunihiro Kaihatsu
  • Total Pages: 3
  • Publisher: Science Impact