The increasing incidence of aspergillosis, a life–threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. Traditional diagnostic methods used in the clinical laboratory include microscopy, culture, or antigen detection. These methods are time-consuming, have low sensitivity and are difficult to standardize. We are developing a rapid real time PCR assay using the LightCycler system for the detection and differentiation of four species of the fungus causing human aspergillosis (Aspergillus fumigatus, A. flavus, A. Niger, and A. terreus). A genus-specific primer set corresponding to the 18s small-subunit rRNA genes was designed. This gene was cloned from A. fumigatus, A. flavus, A. niger, and A. terreus using the TOPO TA Cloning ® kit, (Invitrogen Life Sciences, Carlsbad, CA 92008) and tested with SYBR Green (SG) (LightCycler-FastStart DNA Master SYBR Green 1, Roche Diagnostics GmbH and Roche Molecular Biochemicals, Mannheim, Germany). All aspergillus species were detected by SG melting curves. Other fungal species including Candida albicans, C. tropicalis, C. parapsilosis, C. glabrata, Mucor, Fusarium, Penicillium, Blastomyces dermatitidis, and Absidia species were not amplified. Species-specific capability was obtained by using fluorescence resonance energy transfer (FRET) hybridization probes (LightCycler-FastStart DNA Master Hybridization Probes, Roche Diagnostics GmbH and Roche Molecular Biochemicals, Mannheim, Germany). Probes were designed over a variable region of the genus-specific sequence. Base pair changes under the probe allow the differentiation of each Aspergillus species, resulting in distinctive melting curves. Every base pair mismatch under the probe causes a lowering of the melting temperature. Two sets of the FRET probes have been investigated. They differentiate A. niger from, A. fumigatus, A. terreus and A. flavus. Disappointingly A. fumigatus, A. terreus and A. flavus have the same melting curve. Because of this, new primers and FRET probes are being optimized to differentiate these four clinically significant species of aspergilli. This technology offers promise in the rapid identification of A. fumigatus, A. flavus, A. niger and A. terreus and hopefully for the detection of these fungi in clinical specimens.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)