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Specific detection of fusarium species in blood and tissues by a PCR technique
F X Hue1, M Huerre, M A Rouffault
1Laboratoire de Mycologie Médicale, Institut Pasteur, 75724 Paris cedex 15, France. cneyret@ipagua.gp
Abstract:
Fusarium species are opportunistic nosocomial pathogens that often cause fatal invasive mycoses. We designed a primer pair that amplifies by PCR a fragment of a gene coding for the rRNA of Fusarium species. The DNAs of the main Fusarium species and Neocosmospora vasinfecta but not the DNAs from 11 medically important fungi were amplified by these primers. The lower limit of detection of the PCR system was 10 fg of Fusarium solani DNA by ethidium bromide staining. To test the ability of this PCR system to detect Fusarium DNA in tissues, we developed a mouse model of disseminated fusariosis. Using the PCR, we detected Fusarium DNA in mouse tissues and in spiked human blood. Furthermore, F. solani, Fusarium moniliforme, and Fusarium oxysporum were testing by random amplified polymorphic DNA (RAPD) analysis. The bands produced by RAPD analysis were purified, cloned, and sequenced. The information was used to design primer pairs that selectively amplified one or several Fusarium species. The method developed may be useful for the rapid detection and identification of Fusarium species both from culture and from clinical samples.
Insights
This study developed a PCR method to detect Fusarium species, opportunistic pathogens causing invasive mycoses. The assay accurately identifies Fusarium DNA in clinical samples and tissues, aiding rapid diagnosis.
Area of Science:
- Medical Mycology
- Molecular Biology
- Diagnostic Microbiology
Background:
- Fusarium species are significant causes of fatal invasive mycoses in healthcare settings.
- Accurate and rapid detection of Fusarium is crucial for effective patient management.
Purpose of the Study:
- To develop a Polymerase Chain Reaction (PCR) based method for detecting and identifying Fusarium species.
- To assess the sensitivity and specificity of the developed PCR assay.
- To evaluate the utility of the PCR system in detecting Fusarium DNA in clinical samples and tissues.
Main Methods:
- Design and validation of a primer pair targeting Fusarium ribosomal RNA genes for PCR amplification.
- Determination of the lower limit of detection using Fusarium solani DNA.
- Development of a mouse model for disseminated fusariosis to test the PCR system in vivo.
- Application of Random Amplified Polymorphic DNA (RAPD) analysis for further species differentiation and primer design.
Main Results:
- The designed primers specifically amplified DNA from major Fusarium species and Neocosmospora vasinfecta, but not from 11 other medically important fungi.
- The PCR system demonstrated a high sensitivity, with a lower limit of detection of 10 fg of Fusarium solani DNA.
- Fusarium DNA was successfully detected in mouse tissues and spiked human blood samples.
- RAPD analysis provided information for designing species- or group-specific primer pairs.
Conclusions:
- A sensitive and specific PCR-based method for the detection of Fusarium species has been developed.
- This molecular assay shows promise for the rapid identification of Fusarium from both cultures and clinical specimens.
- The developed technique can significantly aid in the early diagnosis and management of invasive fusariosis.