A molecular detection strategy was developed in Phase I of this project, which has the potential to speed the accurate diagnosis of human disease due to fungal infection. The strategy consists of deriving PCR primers from a conserved region flanking a variable region of the mitochondrial genome. When further developed, this system can solve the challenging problem of accurately and rapidly identifying fungal species and subspecies in the clinical setting. In Phase I, the technical feasibility of the strategy was proved by obtaining discrete, distinctive multiple PCR products from phylogenetically distinct genera and species of fungi. In Phase II, this strategy will be exploited for three applications: 1) identification of Candida species, 2) identification of Aspergillus species, and 3) identification of individual isolates of Aspergillus flavus and A. fumigatus. In Phase II the number of clinical isolates of a given species and the number of target regions will be increased. Primer pairs will be derived that are highly selective. The presence, absence and length of PCR products will be used to establish a species-specific profile. The species.specific, and/or subspecies-specific primer pairs will then be tested in mock specimens using mixed templates of human and fungal DNA to optimize reaction conditions and determine the sensitivity of the assay. Validations of the species.specific and strain-specific profiles on human specimens will be conducted in collaboration with the Department of Laboratory Medicine, University of Washington, Seattle.
One of the major advantages of the polymorphism collection under development in this project is their direct application in detection and identification of fungal pathogens in human specimens. Because of their high sensitivity, high specificity, low costs, and adaptability to automation, the primer collection will be cost effective for medical laboratories and can be available immediately for investigational use.
| Rakeman, Jennifer L; Bui, Uyen; Lafe, Karen et al. (2005) Multilocus DNA sequence comparisons rapidly identify pathogenic molds. J Clin Microbiol 43:3324-33 |
