A sensitive rolling-circle amplification (RCA)-based method utilizing species-specific padlock probes targeted to the internal transcribed spacer 2 region of the fungal ribosomal DNA gene complex was developed. The assay was rapid (2 hours) and specific. Of 28 fungal isolates (16 of Candida, six of Aspergillus, and six of Scedosporium spp.), all were all identified correctly.Fungal pathogens cause life-threatening infections in critically ill and immunosuppressed patients. Contemporary epidemiological trends reveal a shift toward species of Candida and Aspergillus other than Candida albicans and Aspergillus fumigatus and a range of emerging fungi including Scedosporium spp. and the zygomycetes (6,19). Given the reduced susceptibility of many of these pathogens to antifungal agents, timely identification to species level is essential for clinical management. However, standard culture-based identification methods are insensitive and slow (15).To overcome both problems, PCR-based tools have been developed. In particular, the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the fungal ribosomal DNA gene complex have shown promise as targets for species identification in a variety of formats including multiplex and/or real-time PCR assays (9, 16), DNA sequence analysis (1, 2, 12), and probe-based techniques (5, 7). The latter range from Southern blotting (5, 7) and reverse line blot (RLB) hybridization methods (23) to sophisticated microarray formats (10,11,17).Recently, the utility of circularizable oligonucleotide (padlock) probes has been demonstrated for the detection of target nucleic acid sequences, including nucleotide polymorphisms that differ by only a few base pairs, with high sensitivity (4,13,20). Such probes comprise two sequences complementary to the 5Ј and 3Ј termini of the target sequence joined by a linker region (Fig. 1A). Upon hybridization to the target, the probe ends are joined by DNA ligase to form a closed molecule. The intensity of the probespecific signal is then increased exponentially by rolling-circle amplification (RCA) (13) (Fig. 1B). There are few data on the application of padlock probes in the detection of polymorphisms in fungi. We report on a sensitive, RCA-based method using real-time PCR for species identification of clinically important Candida, Aspergillus, and Scedosporium spp.Twenty-eight clinical isolates were studied: two of C. albicans, two of Candida glabrata, three of Candida krusei, three of Candida tropicalis, three of Candida dubliniensis, three of Candida guilliermondii, four of A. fumigatus, two of Aspergillus flavus, and three strains each of Scedosporium apiospermum and Scedosporium prolificans. Species identity was confirmed by standard laboratory methods (3, 21) and ITS sequence analysis (23). Isolates were stored in sterile water at 25°C until required. DNA extraction and amplification of the ITS (ITS1, 5.8S rRNA, and ITS2) region, using the primers ITS1 and ITS4 (22), in preparation for hybridization with padlock probes (see below) were performed a...