Quantitation of Mycelial Fungal Pathogens by PCR

Joel C. Bowman, Jennifer W. Anderson, Paul A. Liberator, Cameron M. Douglas

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Background: Prior studies demonstrated the utility of a quantitative PCR (qPCR) assay for measuring Aspergillus fumigatus tissue burden in murine models of infection. Because filamentous growth produces hyphal masses which cannot accurately be quantified by enumerating colony forming units, we sought to establish qPCR assays for other human fungal pathogens. Methods: Multicopy ribosomal DNA (rDNA) gene sequences from Rhizopus arrhizus, Scedosporium prolificans, and Fusarium solani were used to design oligonucleotide primers and dual-labeled fluorescent probes. Genomic DNA (gDNA) prepared from each strain was analyzed for primer/probe specificity and dynamic range in qPCR reactions (Applied Biosystems). Spores were added to naí¯ve mouse kidneys to establish quantitative detection in tissue. Results: qPCR assays for R. arrhizus, S. prolificans, or F. solani were established with purified gDNA from each organism. There was a direct relationship between DNA concentration and qPCR signal over a 10,000-fold range. Each primer/probe set was selective for the target organism - the qPCR signal detected with gDNA derived from mouse, A. fumigatus, or non-target fungi from this study was at least 106-fold lower than the specific signal. A dilution series of R. oryzae or S. prolificans spores added to naí¯ve kidneys yielded a linear titration over at least 5 orders of magnitude, with a lower limit of detection of 100 spores per gram of kidney. Conclusions: Assays to quantify rDNA from R. oryzae, S. prolificans, or F. solani, either as purified DNA or from tissue-derived samples, have been established. Sensitivity for each assay was consistent with burdens expected in animal models of infection. These qPCR assays should be useful for assessing the progression of infection and for evaluating the efficacy of antifungal therapy.

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Full conference title: 

    • ICAAC 42nd