Rapid identification of pathogenic yeast isolates by real-time PCR and two-dimensional melting-point analysis.

There is a need in the clinical microbiological laboratory for rapid and reliable methods for the universal identification of fungal pathogens. Two different regions of the rDNA gene complex, the highly polymorphic ITS1 and ITS2, were amplified using primers targeting conserved regions of the 18S, 5.8S and 28S genes. After melting-point analysis of the amplified products, the T(m) of the two PCR-products were plotted into a spot diagram where all the 14 tested, clinically relevant yeasts separated with good resolution. Real-time amplification of two separate genes, melting-point analysis and two-dimensional plotting of T(m) data can be used as a broad-range method for the identification of clinical isolates of pathogenic yeast such as Candida and Cryptococcus spp.