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Development of Polymerase Chain Reaction Method for Detection of Human Cytomegalovirus DNA and Its Clinical Application.

Research Project

Project/Area Number 01480261
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field Pediatrics
Research InstitutionSapporo Medical College

Principal Investigator

CHIBA Shunzo  Sapporo Medical College, Department of Pediatrics, Professor, 医学部, 教授 (50045374)

Co-Investigator(Kenkyū-buntansha) SUZUKI Nobuhiro  Sapporo Medical College, Department of Pediatrics, Instructor, 医学部, 助手 (50216420)
YOSHIDA Koichi  Sapporo Medical College, Cancer Institute, Assistant Prof., がん研究所, 講師 (60117653)
YAMANAKA Tatsuru  Sapporo Medical College, Department of Pediatrics. Associate Prof., 医学部, 助教授 (70045508)
Project Period (FY) 1989 – 1990
Project Status Completed (Fiscal Year 1990)
Budget Amount *help
¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1990: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥4,000,000 (Direct Cost: ¥4,000,000)
KeywordsHuman cytomegalovirus / PCR / RT-PCR / DNA hybridization / RTーPCR法 / ドット・ブロット・ハイブリダイゼ-ション
Research Abstract

In order to identify latent state, reactivation and productive infection of human cytomegalovirus (HCMV) at genomic level, polymerase chain reaction (PCR) method and combined reverse transcription-PCR (RT-PCR) method were developed. Two pairs of synthetic oligonucleotide primers coding LA and IE region were designed to amplify the fragment of 305 base pairs from Hind III-V fragment or the fragment of 346 base Paris from Hind III-E fragment of HCMV AD169 strain DNA. PCR was carried out according to the procedure described by Saiki et al (1985). cDNA was synthesized from RNA by reverse transcriptase for performing RT-PCR. Amplified products were detected by gel electrophoresis and by dot blot hybridization with a ^<32>P-labeled oligonucleotide probe. The primers were specific for HCMV strains and did not amplify other herpes family viruses nor human genomic DNA. Reconstitution expriments demonstrated that 10^<-5>pg of the cloned amplified HCMV DNA was detectable by PCR method. IE mRNA was detectable in MRC-5 cells by RT-PCR until 72 hrs after infection with CMV. PCR using a pair of primers coding LA region was proved to be a rapid and sensitive method for detecting HCMV in various clinical specimens including urine and breast milk.
Thus, a combined use of PCR and RT-PCR employing primers of different specificity might provide a useful tool for diagnosing various stages of HCMV infection.

Report

(3 results)
  • 1990 Annual Research Report   Final Research Report Summary
  • 1989 Annual Research Report
  • Research Products

    (11 results)

All Other

All Publications (11 results)

  • [Publications] 母坪 智行 ほか: "Polymerase Chain Reaction法によるヒトサイトメガロウィルスDNA検出法の開発" 札幌医学雑誌. 59. 156-153 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 母坪 智行 ほか: "PCR法によるヒトサイトメガロウイルスDNAの検出" 蛋白質 核酸 酵素・臨時増刊号. 35. 3048-3054 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 母坪 智行 ほか: "DNA増幅法(PCR)の開発とその応用 4)サイトメガロウイルス" 小児科診療・特大号. (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 千葉 峻三: "小児科の進歩11 (小児科学年鑑)" 診療と治療社, (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hotsubo T and Yoshida K: "Detection of human cytomegalovirus DNA by polymerase chain reaction method." Sappro Medical Journal. 59. 153-163 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] Hotsubo T, Chiba S and Fujinaga K: "Detection of human cytomegalovirus DNA by polymerase chain reaction method." Protein, Nucleic Acid and Enzyme. 35. 3048-3054 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1990 Final Research Report Summary
  • [Publications] 母坪 智行ほか: "Polymerase Chain Reaction法によるヒトサイトメガロウイルスDNA検出法の開発" 札幌医学雑誌. 59. 153-163 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 母坪 智行ほか: "PCR法によるヒトサイトメガロウイルスDNAの検出" 蛋白質核酸酵素・臨時増刊号. 35. 3048-3054 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 母坪 智行ほか: "DNA増幅法(PCR)の開発とその応用 4)サイトメガロウイルス" 小児科診療・特大号. (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] 千葉 峻三: "小児科の進歩11(小児科学年鑑)" 診療と治療社, (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] 母坪智行: "Polymerase Chain Reaction法によるヒトサイトメガロウイルスDNA検出法の開発" 札幌医学雑誌. 59. (1990)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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