Simultaneous Detection and Identification of Candida , Aspergillus , and Cryptococcus Species by Reverse Line Blot Hybridization

Abstract

We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit crosshybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10 0.5 cells/ml and 10 2 conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.

Simultaneous Detection and Identification of Aspergillus and Mucorales Species in Tissues Collected from Patients with Fungal Rhinosinusitis

“…For nine specimens with a diagnosis of chronic 1 to 10 2 conidia/ml in normal saline. This is similar to the sensitivity of RLB observed in other studies (16,23). Further investigations using 10-fold serial dilutions of genomic DNA of A. fumigatus indicated that the minimum concentration that can be detected by the PCR/RLB assay is 1.8 ϫ 10…”

“…To allow optimal hybridization under the same conditions, probes were also designed to have similar physical characteristics: lengths between 20 and 28 bp and melting temperatures between 54.48°C and 63.68°C (Table 2). Oligonucleotide probes for Aspergillus species were used as described previously or modified from these previously described probes (16,23). Mucorales speciesspecific probes were designed by the authors, targeting ITS1 or ITS2 sequences ( Table 2).…”

“…For the clinical specimens, the QIAamp DNA FFPE tissue kit (Qiagen, Germany) and the QIAamp DNA minikit (Qiagen, Germany) were used to extract DNA from the PE and surgical tissues, respectively. Control of contamination was performed as described before, e.g., specimen manipulations and DNA extractions were performed in a class II laminar flow cabinet (16,23).…”

“…Relevant fungal DNA sequences spanning the fungal ribosomal DNA gene complex (ITS1, 5.8S rRNA, and ITS2) were accessed from GenBank and compared using the DNAMAN software program (Lynnon Biosoft). Three pairs of 5Ј-end biotin-labeled primers (Sangon, China), based on the universal fungal primer pairs ITS1 and ITS4, ITS1 and ITS2, and ITS3 and ITS4 (7,16,23), were used for the amplification of the ITS, ITS1, and/or ITS2 region (Table 2). To allow optimal hybridization under the same conditions, probes were also designed to have similar physical characteristics: lengths between 20 and 28 bp and melting temperatures between 54.48°C and 63.68°C (Table 2).…”

New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization

“…Recently, the use of oligonucleotide probes in reverse line blot (RLB) hybridization assays proved to be powerful for the identification of a diverse range of clinically relevant human and veterinary pathogens, with high throughput (14,40,42,44,54). Briefly, the RLB method enables the nonradioactive hybridization of PCR amplicons with different oligonucleotide probes in a single assay.…”