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In-situ detection of Aspergillus fumigatus
Ryo Hanazawa1, Somay Yamagata Murayama1, Hideyo Yamaguchi1
1*Department of Microbiology and Immunology, Teikyo University School of Medicine, 2-11-1, Kaga, Itabashi-Ku, Tokyo 173-8605 and †Teikyo University Institute of Medical Mycology, 359, Otsuka, Hachioji, Tokyo 192-0395, Japan.
Abstract:
An in-situ hybridisation (ISH) technique to detect Aspergillus fumigatus in infected tissues was developed in which 568-bp, 333-bp and 154-bp PCR products of the alkaline proteinase gene were employed. Dot-blot hybridisation with the 568-bp probe on a membrane containing genomic DNA from several different fungi including A. flavus, A. niger, Penicillium spp., Mucor racemosus or Pseudallescheria boydii gave negative results. ISH was done on formalin-fixed, paraffin-embedded pulmonary tissues from rats infected with A. fumigatus and renal tissues from mice infected with A. fumigatus, A. flavus or A. niger. The 568-bp probe reacted strongly in ISH with both A. fumigatus and A. flavus, and weakly with A. niger. The 333-bp probe also reacted in ISH with A. fumigatus and A. flavus, although the intensity was weaker. However, in ISH with the 154-bp probe, there was no positive signal with any Aspergillus spp. These results demonstrate that A. fumigatus and A. flavus can be specifically detected in infected tissues by ISH with the 568-bp probe. This technique could be applicable to clinical specimens for molecular diagnosis of aspergillus infections.
Insights
A new in-situ hybridisation (ISH) technique accurately detects Aspergillus fumigatus in infected tissues. This molecular diagnostic method shows promise for identifying fungal infections in clinical specimens.
Area of Science:
- Mycology
- Molecular Biology
- Pathology
Background:
- Aspergillus fumigatus is a significant cause of invasive fungal infections.
- Accurate and specific detection methods are crucial for diagnosing aspergillosis.
- Existing diagnostic techniques may lack specificity or sensitivity.
Purpose of the Study:
- To develop and validate an in-situ hybridisation (ISH) technique for detecting Aspergillus fumigatus in infected tissues.
- To assess the specificity of the developed ISH probes against other fungal species.
- To evaluate the applicability of the ISH technique for molecular diagnosis of aspergillus infections.
Main Methods:
- Development of polymerase chain reaction (PCR) products of the alkaline proteinase gene from Aspergillus fumigatus (568-bp, 333-bp, and 154-bp).
- Dot-blot hybridisation to assess probe specificity against various fungal DNA.
- In-situ hybridisation (ISH) on infected rat and mouse tissues using the developed probes.
Main Results:
- Dot-blot hybridisation confirmed the specificity of the 568-bp probe against non-Aspergillus fungi.
- ISH with the 568-bp probe showed strong reactivity with Aspergillus fumigatus and Aspergillus flavus, and weak reactivity with Aspergillus niger.
- The 333-bp probe showed weaker reactivity with Aspergillus fumigatus and Aspergillus flavus, while the 154-bp probe yielded no positive signals.
Conclusions:
- The 568-bp probe enables specific detection of Aspergillus fumigatus and Aspergillus flavus in infected tissues via ISH.
- The developed ISH technique is a promising tool for the molecular diagnosis of aspergillus infections in clinical settings.