Identification of human pathogenic fungi via DNA-Microarray analysis for clinical applications
Patients with a weak immune system like people receiving immuno-suppressive treatment for cancer and organ transplantation or patients who suffer from AIDS or cystic fibrosis are representing a high-risk group for secondary infections with human pathogenic fungi. Those invasive fungal infections show a high morbidity and mortality rate between thirty to eighty percent. Deciding reasons may be inadequate medication due to inaccurate and time consuming classification of moulds and yeasts in clinical laboratories. For an increased life expectancy, an effective and early medication is necessary. Conventional molecular biological methods to identify human pathogenic species like PCR, qRT-PCR or sequencing preclude parallel detection resulting in material and sample intensity. To overcome these limitations we are developing a Fungal-Yeast-Identification-Chip as a fast and reliable device for the accurate identification of 55human pathogenic moulds and yeasts. To this end we take advantage of DNA sequences of specific target genes which are representing evolutionarily conserved sequences variable enough to discriminate the relevant species. Sequence databases of ribosomal RNA genes as well as functional target genes are established to design probes with high discrimination power and primer pairs for the amplification of diagnostic target regions. To evaluate the DNA-Microarray in a first step individual PCRs with DNA of reference species were established. Up to now evaluation of the microarray has been completed with 55 reference species. After hybridization of labeled amplicons highly specific signals were obtained. Background fluorescence signals are very low and could be discounted. To increase specific signals of designed probes a probe-redesign took place. Furthermore, the microarray will be validated with patient samples like broncho-alveolar lavage or tracheal secretion provided by our clinical partner. For clinical application the Fungal-Yeast-Identification-Chip will be embedded in a Lab-on-a-chip-system. All required steps like cell lysis of human and pathogenic cells, amplification of target genes via PCR and the identification of 55 fungal pathogens via microarray are integrated in a disposable cartridge. Reliable and fast identification should be possible within a few hours and provides rapid therapeutic intervention in an early state of infection.