In:
Journal of Clinical Microbiology, American Society for Microbiology, Vol. 38, No. 10 ( 2000-10), p. 3735-3742
Abstract:
We report on a reverse-hybridization line probe assay (LiPA) which when combined with PCR amplification detects and identifies clinically significant fungal pathogens including Candida , Aspergillus , and Cryptococcus species. DNA probes have been designed from the internal transcribed-spacer (ITS) regions of Candida albicans , Candida parapsilosis , Candida glabrata , Candida tropicalis , Candida krusei , Candida dubliniensis , Cryptococcus neoformans , Aspergillus fumigatus , Aspergillus versicolor , Aspergillus nidulans and Aspergillus flavus . The probes were incorporated into a LiPA for detection of biotinylated ITS PCR products, and the specificity of the probes was evaluated. We established LiPA detection limits for ITS 1 and for full ITS amplicons for genomic DNA from C. albicans , A. fumigatus , and C. neoformans . Further evaluation of the LiPA was carried out on clinical fungal isolates. One hundred twenty-seven isolates consisting of dimorphic yeasts and dermatophytic and filamentous fungi were tested by the LiPA, which correctly identified 77 dimorphic yeasts and 23 of the filamentous isolates; the remaining 27 isolates represented species of fungi for which probes were not included in the LiPA. The fungal-PCR-LiPA technology was applied to blood samples inoculated with Candida cells which were pretreated by minibead beating to mechanically disrupt the cells, with the DNA extracted by either a previously described guanidium thiocyanate-silica method or the commercially available QIAmp tissue kit. PCR amplification of the extracted DNA and subsequent DNA probe hybridization in the LiPA assay yielded detection limits of 2 to 10 cells/ml. An internal standard control was included in the PCR amplification to monitor for PCR inhibition. This fungal PCR-LiPA assay is robust and sensitive and can easily be integrated into a clinical-testing laboratory with the potential for same-day diagnosis of fungal infection.
Type of Medium:
Online Resource
ISSN:
0095-1137
,
1098-660X
DOI:
10.1128/JCM.38.10.3735-3742.2000
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2000
detail.hit.zdb_id:
1498353-9
SSG:
12
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