Development of a universal system for fungal species identification and SNP typing via onchip minisequencing

Fungal infections are a predominant clinical problem, especially in intensive care units. In particular for patients with a defective immune system, fungal infections are associated with high mortality rates. Nevertheless, a fast and well directed medication may improve patient outcome significantly. A further problem is the increasing resistance against the leading antimycotics due to the rise of inherently resistant fungal species or during long term treatment. The resistance mechanisms of fungal pathogens are often based on single nucleotide polymorphisms (SNPs) in genes regulating the expression of pumps extruding the drug outside the fungal cell or encoding for the target of the antimycotica. Since development of resistance is not predictable, constant resistance monitoring is necessary to enable adequate patient medication. In the present study we developed a system for the highly parallel detection of fungal species and their SNPs associated with azole resistance using an on-chip minisequencing technology. Minisequencing allows parallel analysis of SNPs both in homo- and heterozygous strains and offers a good platform in terms of species identification. For the minisequencing reaction the spotted probes are hybridized with PCR products from clinical samples and synthetic control probes. In the enzymatic reaction, different fluorescently labelled dideoxynucleotides and a thermo stable sequenase are used for the specific extension of the probes with the perfectly matching nucleotide. Based on this system, we developed a prototype chip with 15 species specific probes for Candida albicans, C. glabrata and Aspergillus fumigatus based on ITS or 18s rRNA sequences as well as SNP probes for erg11, tac1 and mrr1 SNPs of C. albicans from our existing SNP chip. Mutations in these genes are central for causing resistance. Furthermore, four control probes used to confirm the correct incorporation of the fluorescently labelled didesoxynucleotides have been developed to enable normalisation, which is essential for correct identification of heterozygosity. The chip has been successfully validated with synthetic templates and with defined PCR products from clinical isolates. In the future, the chip will be expanded by further resistance associated SNPs and by onchip minisequencing based species identification probes. This work has been financially supported by the EURESFUN project (EU-FP6-STREP).