Development of diagnostic method for comprehensive viral detection using not so random primer sets

• Project/Area Number: 25670271
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Laboratory medicine
• Research Institution: Osaka University
• Principal Investigator: NAKAMURA SHOTA 大阪大学, 微生物病研究所, 助教 (90432434)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2014: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2013: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
• Keywords: 感染症 / メタゲノム / 応用ゲノム科学 / 臨床検査学 / 微生物学 / ウイルス学 / バイオインフォマティクス

Outline of Final Research Achievements

Unbiased next-generation sequencing approaches enable a diagnostic method for comprehensive pathogen detection. This method employs a single common protocol without specific treatment for each pathogen, which consists of the amplification of DNA/RNA in clinical specimens by a random primer set, metagenomic shotgun sequencing, and identification of pathogenic genes. Our previous studies have revealed that a large amount of human genomic DNA in clinical specimens decreases the sensitivity for pathogen detection. Therefore, we tried to develop the subtraction method for human-derived sequences using not so random primer sets. Using the newly designed primer sets, we were able to subtract the human sequences and amplify the target viral sequences from the specimen of influenza A infection. However, undesirable environmental bacterial sequences were obtained coincidentally. Further study on improving the sensitivity by redesign of the primer sets and protocols needs to be considered.

Research Products

• [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] 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] Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes. (2014)
  • Author(s): Miyamoto M, Motooka D, Gotoh K, Imai T, Yoshitake K, Goto N, Iida T, Yasunaga T, Horii T, Arakawa K, Kasahara M, Nakamura S.
  • Journal Title: BMC Genomics Volume: 15 Issue: 1 Pages: 699-699
  • DOI: 10.1186/1471-2164-15-699
  • Peer Reviewed / Open Access
• [Journal Article] Comprehensive metagenomic approach for detecting causative microorganisms in culture-negative infective endocarditis. (2014)
  • Author(s): Imai A, Gotoh K, Asano Y* (corresponding author), Yamada N, Motooka D, Fukushima M, Kanzaki M, Ohtani T, Sakata Y, Nishi H, Toda K, Sawa Y, Komuro I, Horii T, Iida T, Nakamura S, Takashima S.
  • Journal Title: Int J Cardiol. Volume: 172 Issue: 2 Pages: 288-9
  • DOI: 10.1016/j.ijcard.2013.12.197
  • Peer Reviewed
• [Journal Article] Virus purification and enrichment by hydroxyapatite chromatography on a chip (2014)
  • Author(s): Niimi, M., Masuda, T., Kaihatsu, K., Kato, N., Nakamura, S., Nakaya, T., and Arai, F.
  • Journal Title: Sensors and Actuators B: Chemical Volume: 201 Pages: 185-190
  • Peer Reviewed
• [Journal Article] Allergic bronchopulmonary mycosis due to co-infection with Aspergillus fumigatus and Schizophyllum commune (2014)
  • Author(s): Seki, M., Ohno, H., Gotoh, K., Motooka, D., Nakamura, S., Iida, T., Miyazaki, Y., and Tomono, K.
  • Journal Title: ID Cases Volume: 1 Pages: 5-8
  • Peer Reviewed
• [Journal Article] Severe respiratory failure due to co-infection with human metapneumovirus and Streptococcus pneumoniae (2014)
  • Author(s): Seki, M., Yoshida, H., Gotoh, K., Hamada, N., Motooka, D., Nakamura, S., Yamamoto, N., Hamaguchi, S., Akeda, Y., Watanabe, H., Iida, T., and Tomono, K.
  • Journal Title: Respiratory Medicine Case Reports Volume: 12 Pages: 13-15
  • Peer Reviewed
• [Presentation] 次世代シークエンスの過去・現在・未来～ウィルス研究に与えるインパクト (2014)
  • Author(s): 中村 昇太
  • Organizer: 第62回 日本ウィルス学会学術集会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2014-11-11
  • Invited
• [Presentation] Genomics and Metagenomics using 2nd and 3rd Generation Sequencers (2014)
  • Author(s): 中村 昇太
  • Organizer: DMSc-NIH Seminar
  • Place of Presentation: ノンタブリ（タイ）
  • Year and Date: 2014-07-03
• [Book] 次世代シークエンス解析スタンダード NGSのポテンシャルを活かしきるWET&DRY (2014)
  • Author(s): 二階堂 愛、中村 昇太 他
  • Total Pages: 401
  • Publisher: 羊土社