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Development of a chromosomal DNA probe for the laboratory diagnosis of aspergillosis
1Department of Microbiology & Preventive Medicine, Iowa State University, Ames 50011.
Abstract:
Chromosomal DNA was extracted from clinical isolates of Aspergillus fumigatus of human and animal origin using the protoplast lysate method. The probe was developed by the nick translation of the chromosomal DNA genome fraction with p32 as the radiolabel. Hybridization of the probe with endonuclease-cleaved DNA of the same species resulted in a pattern of recognition sites specific for the species. The latter was not seen in other species encountered in clinical specimens. Trials were carried out on sputum experimentally inoculated with the fungus where crude DNA was directly extracted, treated with the endonuclease and hybridized with the probe. The efficacy of the probe was as good with the crude as the purified DNA. The specificity of the probe was determined by testing it against single and mixed DNA populations extracted from different species of several fungal and bacterial genera isolated from and/or known to occur in clinical specimens of respiratory infection origin. The sensitivity of the probe was assessed by detecting a DNA concentration in the specimen equivalent to 3 C.F.U.
Insights
A new DNA probe accurately identifies Aspergillus fumigatus in clinical samples. This probe works effectively with both crude and purified DNA, offering a specific and sensitive diagnostic tool for respiratory infections.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Nucleic Acid Hybridization
Background:
- Aspergillus fumigatus is a significant opportunistic pathogen causing invasive fungal infections.
- Accurate and rapid identification of A. fumigatus is crucial for effective patient management.
- Current diagnostic methods can be slow or lack specificity.
Purpose of the Study:
- To develop and evaluate a species-specific DNA probe for the identification of Aspergillus fumigatus.
- To assess the probe's efficacy using clinical isolates from human and animal sources.
- To determine the probe's sensitivity and specificity in detecting A. fumigatus in respiratory specimens.
Main Methods:
- Chromosomal DNA extraction from clinical isolates of A. fumigatus using the protoplast lysate method.
- Development of a radiolabeled DNA probe (using p32) via nick translation.
- Hybridization assays with endonuclease-cleaved DNA from various fungal and bacterial species.
- Testing the probe with crude and purified DNA from experimentally inoculated sputum samples.
Main Results:
- The developed probe demonstrated specific recognition patterns for A. fumigatus.
- No cross-reactivity was observed with other fungal or bacterial species commonly found in clinical specimens.
- The probe showed comparable efficacy with both crude and purified DNA.
- Sensitivity was sufficient to detect DNA equivalent to 3 colony-forming units (CFU).
Conclusions:
- A species-specific DNA probe for Aspergillus fumigatus has been successfully developed.
- The probe is effective and specific for identifying A. fumigatus in clinical samples.
- This molecular tool holds promise for improved diagnostics of A. fumigatus infections.