| Project/Area Number |
16590295
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Human pathology
|
|---|
| Research Institution | Kitasato University |
|---|
Principal Investigator |
MURAYAMA Somay Kitasato University, Kitasato Institute for Life Sciences, Assistant Professor, 北里生命科学研究所, 講師 (60183654)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
UBUKATA Kimiko Kitasato University, Kitasato Institute for Life Sciences, Professor, 北里生命科学研究所, 教授 (70082302)
|
|---|
| Project Period (FY) |
2004 – 2005
|
| Project Status |
Completed (Fiscal Year 2005)
|
| Budget Amount *help |
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2005: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
|
|---|
| Keywords | Aspergillus fumigatus / Aspergillus flavus / Aspergillus niger / in situ hybridization / molecular diagnosis / pathogenic fungi / tissue / probe / Aspergillus / A. fumigatus / A. flavus / ISH / hybridization / 真菌同定 / A.fumigatus / A.flavus / 真菌 / 同定 |
|---|
| Research Abstract |
A novel in situ hybridization (ISH) technique was developed to detect and diagnose Aspergillus fumigatus, a major pathogen of opportunistic fungal infection. In this ISH procedure, we employed the 568 bp-probe derived from the A.fumigatus alkaline proteinase gene (ALP). The species-specificity of this probe was confirmed by a dot-blot hybridization performed on a membrane applying various genomic DNAs prepared from several related or different fungi including A.flavus, A.niger, Penicillium spp., Mucor racemosus, Pseudallescheria boydii and Candida albicans, as well as A.fumigatus. An ISH which was done with the 568bp-probe on formalin fixed, paraffin embedded pulmonary tissues of rats infected with A.fumigatus under optimal assay conditions gave clear and plentiful signals in the pulmonary lesions where fungal elements were demonstrated to exist by PAS stain. Two shorter probes, the 333 bp-probe and the 154 bp-probe, were also tested for their efficiency in ISH. In comparison with the signals obtained with the 568 bp-probe, those with the 333 bp-probe were weaker in intensity and there were virtually none with the 154 bp-probe. Based on these results, our ISH technique with the 568 bp-probe is useful in detecting an identifying A.fumigatus histopathologically in the tissue specimens and expected to be clinically applicable as a new molecular diagnostic method for aspergillosis.
We also designed A.flavus specific probe within its retrotransposable element Afut1. We have chosen the long terminal repeat (LTR) region. The 225 bp-probe showed positive signal with A.flavus infected tissue and slightly reacted with A.nidulans infected tissue. Therefore we newly designed peptide nucleic acid (PNA) probe and locked nucleic acid (LNA) probe. Both probe shows strong specificity and sensitivity.
|
