| Project/Area Number |
06557020
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | University of Tokushima |
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Principal Investigator |
OHNISHI Yoshinari University of Tokushima, School of medicine, professor., 医学部, 教授 (10037400)
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| Co-Investigator(Kenkyū-buntansha) |
KAMOGASHIRA Takashi Otsuka Pharmaceutical Co., Ltd., New Product Evaluation and Development, vice di, 新薬開発部, 部長補佐
AKIMOTO Shigeru University of Tokushima, School of medicine, associate professor., 医学部, 助教授 (10159337)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1994: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | Bacteroides fragilis / DNA probe / PCR / Rapid detection method / Neuraminidase / DNA diagnosis / Infection / rRNA spacer region |
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| Research Abstract |
Oligonucleotide primers and a DNA probe were designed on the basis of the sequence of the neuraminidase-encoding gene (nanH) of Bacteroides fragilis and used for the specific detection of this anaerobe by the nested PCR and hybridization assay. The detection limits of B.fragilis cells and DNA by the nested PCR assay were 10 colony forming units and 10 fg of chromosomal DNA,respectively. To determine the specificity of the first and second primer sets, 59 strains of B.fragilis and 45 strains of other species were tested. All strains of B.fragilis produced the DNA fragment of correct size by PCR using each primer set. Other bacterial species tested were all negative by either of the primer sets, except for a strain of B.vulgatus, which produced a DNA fragment close in size to that of B.fragilis with the second primer set. The dot blot hybridization assay using a 3'-digoxigenin-labeled oligonucleotide probe, PH1, was able to exclude the false-positive result. Positive spots in the hybridi
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zation assay were detected from the PCR products of all 59 strains of B.fragilis, while no positive spots were detected from other bacterial species. The nested PCR-dot blot hybridization assay was performed using blood samples from a sepsis-model mouse of B.fragilis infection. Twenty colony forming units of B.fragilis in 10mul of the blood could be detected by this assay. The nested PCR-dot blot hybridization assay targeting the nanH gene is expected to be applied to clinical specimens from patients with bacteremia as a rapid detection system for B.fragilis.
We purified a fibrinogen-degrading protease from B.fragilis. Cloning and sequencing of the protease-encoding gene will provide another target that can reduce false negative results when used in combination with nanH.Moreover, we determined the nucleotide sequence of beta-isopropylmalate dehydrogenase gene (leuB) of B.fragilis and the sizes of 16S-23S rRNA intergenic spacer regions in seven Bacteroides species including B.fragilis. These results will be useful for future studies to develop a system for differentiation of Bacteroides species. Less
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