Practical Method for Detection and Identification of Candida, Aspergillus , and Scedosporium spp. by Use of Rolling-Circle Amplification

Abstract

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...

Identification and Typing of Isolates of Cyphellophora and Relatives by Use of Amplified Fragment Length Polymorphism and Rolling Circle Amplification

“…To increase its discriminative specificity, the 3=-end binding arm was designed with a T m of 10 to 15°C below ligation temperature. The linker regions of each Cyphellophora species-specific probe were taken from Zhou et al (23), and the 5= and 3= binding arms were designed in this article ( Table 2). Sequences of the two primers used for RCA and the oligonucleotide padlock probes are listed in Table 2.…”

“…Subsequently, rolling circle amplification (RCA) is developed as a sensitive, rapid, and costeffective technique to identify the cutaneous Cyphellophora and Phialophora species. RCA is an isothermal amplification method which has been applied for molecular diagnosis of microbial human pathogens (21)(22)(23). We developed seven RCA padlock probes for the main species in the clade of cutaneous agents of Cyphellophora and Phialophora.…”

Development and Validation of a Microsphere-Based Luminex Assay for Rapid Identification of Clinically Relevant Aspergilli

“…Over the last several years, molecular methods, including the rolling-cycle amplification, repetitive sequence-based PCR, PCR-restriction enzyme, reverse line blot assay, and DNA microarray methods, have been evaluated for Aspergillus species identification (7,14,19,22,25). Although these methods have been demonstrated to be useful for species identification, most of these methods (except the reverse line blot assay) are not amenable to multiplexing.…”

Development and Validation of a Multiplex PCR for Detection of Scedosporium spp. in Respiratory Tract Specimens from Patients with Cystic Fibrosis

“…An ITS-directed pan-fungal PCR assay combined with DNA sequencing was used to detect multiple fungal genera, including S. prolificans, in fresh and formalinfixed clinical specimens (19). Recently, multiplex-tandem PCR (MT-PCR)-and rolling circle amplification (RCA)-based approaches successfully identified S. prolificans and S. apiospermum sensu lato from isolated colonies (both methods) and from blood cultures (MT-PCR) (20,21,29). However, most of these assays do not take into account the taxonomic reclassi-fication of Scedosporium spp.…”

“…in clinical specimens, however, are insensitive and time-consuming, as culture from sputum samples may require up to 14 days to produce fungal growth adequate for morphological identification (1,5). A number of molecular detection and identification methods for Pseudallescheria/Scedosporium have been reported (3,4,19,20,21,29). Real-time PCR protocols for detection of S. prolificans and S. apiospermum sensu lato have been developed (4).…”