| Project/Area Number |
10671646
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Ophthalmology
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| Research Institution | The University of Tokushima |
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Principal Investigator |
SHIOTA Hiroshi The University of Tokushima, School of Medicine, Professor, 医学部, 教授 (20035736)
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| Co-Investigator(Kenkyū-buntansha) |
KAMADA Yasuo The University of Tokushima, School of Medicine, Research Associate, 医学部・附属病院, 助手 (20304529)
KAJIMA Makoto The University of Tokushima, School of Medicine, Research Associate, 医学部, 助手 (90294678)
KUDO Eiji The University of Tokushima, School of Medicine, Assistant Professor, 医学部・附属病院, 講師 (50274220)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1999: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1998: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | herpes simplex virus / HSV / herpetic keratitis / thymidine kinase / DNA polymerase / drug resistance / PCR-SSCP |
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| Research Abstract |
Herpetic keratitis is a corneal disease due to the infection of herpes simplex virus(HSV). Difficult herpetic keratitis to deal with is often caused by gene mutations of thymidine kinase(TK) and/or DNA polymerase . A new quick method to detect these mutations was developed.
1) Design of primers : The TK gene was consisted of 1131 base pairs. Seven primers to cover these pairs were designed and verified to be effective in detecting mutations of the TK gene. Meanwhile, the DNA polymerase gene was consisted of 3708 base pairs. Among these codon 437-963 have keys for drug-resistance. Seven primers to cover the codon were designed and found to be effective in detecting mutations of DNA polymease gene.
2) Establishment of drug-resistant HSV and the analysis of its mechanism : PH strain of HSV-1 was passaged 10 times in three different media which contain low concentration of carbocyclic oxetanocin G(C. OXT-G) or adenine arabinoside (ara-A) or foscarnet. A C.OXT-G resistant HSV was established and its genetic analysis was done using the above primers and found that it was caused by a deletion of guanine at 430 in the TK gene, whilst no mutations were found in the DNA polymerase gene. Resistant strains were not established by the use of ara-A nor foscarnet.
Mutations of TK and/or DNA polymerase could be detected rather quickly by the use of our primers and a combination of polymerase chain reation-single strand conformation polymorphism and direct sequencing.
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