To validate the functionality of a previously described molecular detection method for fungal pathogens, a real-time PCR assay targeting the 18S rRNA gene was used to detect DNA from clinically significant filamentous fungi, (Loeffler, et al, 2000. JCM 38:586-590). Nucleic acid from ATCC strains and clinical isolates was extracted using PrepMan™ Ultra (Applied Biosystems, Foster City, CA) under standard conditions. Published universal primers targeting consensus sequences in the 18S region were used to amplify DNA from the following species: Aspergillus flavus (n=5), A. fumigatus (n=5), A. glaucus (n=4), A. nidulans (n=4), A. niger (n=5), A. terreus (n=5), A. versicolor (n=3), Blastomyces dermatitidis (n=5), Coccidioides immitis (n=5), and Histoplasma capsulatum (n=5). The following genera were also included: Absidia (n=3), Conidiobolus (n=1), Cunninghamella (n=1), Mucor (n=3), Rhizopus (n=4), Rhizomucor (n=2), and Saksenaea (n=2). Amplification was performed using commercially available reagents (LightCycler-FastStart DNA Master Hybridization Probes and LightCycler-FastStart DNA Master SYBR Green 1, Roche Diagnostics GmbH and Roche Molecular Biochemicals, Mannheim, Germany) and LightCycler technology. DNA amplification was measured by SYBR green fluorescence (SG), and appropriate melting curves were observed with all tested species except Rhizomucor. 18S fluorescence resonance energy transfer (FRET) of the probes was observed from all species of Aspergillus, Blastomyces, and Coccidioides included in this study. Significant cross-reactivity between Aspergillus flavus, A. fumigatus, A. glaucus, A. nidulans, A. niger, A. terreus, and A. versicolor was also noted. Several clinical isolates of Rhizopus and Absidia could be delineated based on FRET melting curve analysis. Amplified DNA from the majority of other zygomycetes could not be detected with the FRET, but were detected with SG. In an ongoing prospective clinical study, bronchoalveolar lavage fluid [n=24] were extracted using MagNA Pure LC Total Nucleic Acid Isolation Kit-Large Volume and S.T.A.R. (Roche Diagnostics Gmb H and Roche Molecular Biochemicals, Mannheim, Germany), amplified by FRET and SG, and compared to culture. All samples were FRET negative. However, SG was positive in all samples. To date, 19 cultures have been finalized with 9 negative and the others positive for A. flavus (n=2), C. albicans, (n=4), Penicillium spp. (2), S. cerevisiae and C. glabrata. Additional studies are underway utilizing primers and probes based on other fungal target sequences. These studies show promise in clinical application of real-time PCR for rapid and specific identification of pathogenic fungi.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)