| Project/Area Number |
11670404
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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 |
Public health/Health science
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| Research Institution | Osaka Prefectural Institute of Public Health |
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Principal Investigator |
KATSUKAWA Chihiro Osaka Prefectural Institute of Public Health, Department of Public Health, Senior Researcher, 公衆衛生部, 主任研究員 (20183725)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Yasuhiko Osaka Prefectural Institute of Public Health, Department of Public Health, Senior Researcher, 公衆衛生部, 主任研究員 (90206540)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2000: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1999: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Mycobacterium tuberculosis / Pyrazinamide / drug susceptibility testing / pyrazinamidase / pncA gene / gene mutation / rapid detection method / in vitro transcription-translation coupled system / 翻訳システム / pncA / 耐性 / ダイレクトシークエンス / 多剤耐性結核菌 / 小川培地 / 液体培地 |
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| Research Abstract |
Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug for tuberculosis which requires conversion to the bactericidal compound pyrazinoic acid by bacterial pyrazinamidase activity. Mutations leading to a loss of pyrazinamidaseactivity cause PZA resistance in Mycobacterium tuberculosis. Thus, the detection of pyrazinamidase activity makes the discrimination of PZA resistant tuberculosis possible. However, the detection of the pyrazinamidase activity of M. tuberculosis isolates needs a large amount of bacilli and is therefore time consuming. In this study, we developed a new method for the detection of pyrazinamidase activity with a PCR-based system. The genes encoding pyrazinamidase (pncA genes) in 30 resistant clinical isolates were amplified by PCR by using forward primers containing bacteriophage T7 promoter sequences at the 5' ends. Then the PCR products were directly subjected to an in vitro transcription-translation coupled system. All of the PZA-resistant isolates tested showed reduced pyrazinamidase activity compared to susceptible M. tuberculosis type strain H37Rv. In contrast, all of the 15 susceptible clinical isolates exhibited pyrazinamidase activities similar to that of H37Rv. This fact suggested the possibility of the usefulness of this system for the rapid detection of PZA-resistant M. tuberculosis.
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