DNA-Microarray for Fungal Species Identification and Monitoring of Resistance-Associated SNPs in Candida albicans
The present scientific work describes the development of a microarray-based, diagnostic device for both, the identification of clinically relevant human-pathogen fungi, and the monitoring of resistance-associated SNPs (Single Nucleotide Polymorphisms) in C. albicans. For this purpose the APEX-Method (Arrayed Primer Extension Reaction) was chosen, as it allows combining both platforms in one device. All APEX-probes have been successfully validated with synthetic templates and were tested with clinical samples. To overcome the problem of overlapping emission and extinction spectra of fluorescence dyes, a method was developed to mathematically correct the false positive signal resulting from the dye cross-talk of cy 3 and fluorescein. For an application as a diagnostic device, the experimental protocol has been streamlined to 6 hours, starting from extracting DNA. In addition, for epidemiological purposes, the SNP screening was compared to conventional MLST sequencing analysis in order to correlate the clustering of SNPs related to resistance with a clade association. Taken together, this work clearly demonstrates the applicability of three dyes for sequencing of DNA at reasonable cost and within acceptable process time managing the influence of crosstalk by mathematical reduction of the respective coefficient.