Detection and identification of fungi in blood using broad-range 28S rDNA PCR amplification and species-specific hybridisation

The aim of the present study was to develop a PCR-based method to detect and identify fungi directly from human venous blood. We used broad-range PCR primers that targeted a part of the large subunit 28S rRNA genes. To obtain species-specific hybridisation probes, type strains of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, C. tropicalis and Cryptococcus neoformans were PCR amplified, and the amplicons were analysed by gene sequencing. Based on the sequence analysis, species-specific probes that targeted variable regions were designed and used in hybridisation analyses. Between 2 to 10 fungal cells/ml of spiked blood samples could be detected and correctly identified to species. We applied the technique to blood samples obtained from two patients with or two patients without verified candidaemia. The three samples of candidaemia patients were correctly identified to species level, and those of the negative patients remained negative. This method is a potential tool for diagnosis of systemic invasive candidiasis.