2008
DOI: 10.1128/jcm.00420-08
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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 r… Show more

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Cited by 65 publications
(73 citation statements)
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“…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.…”
Section: Isolatesmentioning
confidence: 99%
See 1 more Smart Citation
“…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.…”
Section: Isolatesmentioning
confidence: 99%
“…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.…”
mentioning
confidence: 99%
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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.…”
mentioning
confidence: 99%
“…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).…”
mentioning
confidence: 99%