title: Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system.
abstract: The Light Cycler technique combines rapid in vitro amplification of DNA in glass capillaries with real-time species determination and quantification of DNA load. We have established a quantitative PCR protocol for two clinically important pathogens, Candida albicans and Aspergillus fumigatus. The sensitivity of the assay was comparable to those of previously described PCR protocols (5 CFU/ml). Specific detection of C. albicans and A. fumigatus could be achieved. The assay showed a high reproducibility of 96 to 99%. The assay was linear in a range between 10(1) and 10(4) Aspergillus conidia. As capillaries do not have to be reopened for post-PCR analysis, the risk of carryover contaminations could be minimized. The Light Cycler allowed quantification of the fungal loads in a limited number of clinical specimens from patients with hematological malignancies and histologically proven invasive fungal infections. Five of nine positive samples had fungal loads between 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads between 10 and 100 CFU/ml of blood, and two of nine samples had fungal loads of more than 100 CFU/ml of blood. All samples were also found to be PCR positive by PCR-enzyme-linked immunosorbent assay analysis.
authors.affiliation: Medizinische Klinik, Abteilung II, Eberhard-Karls-Universität, 72076 Tübingen, Germany. juergen.loeffler@med.uni-tuebingen.de
authors.forename: J
authors.initials: J
authors.lastname: Loeffler
authors.forename: N
authors.initials: N
authors.lastname: Henke
authors.forename: H
authors.initials: H
authors.lastname: Hebart
authors.forename: D
authors.initials: D
authors.lastname: Schmidt
authors.forename: L
authors.initials: L
authors.lastname: Hagmeyer
authors.forename: U
authors.initials: U
authors.lastname: Schumacher
authors.forename: H
authors.initials: H
authors.lastname: Einsele
mesh_terms: D001228:Aspergillosis / Q000175:diagnosis* / Q000382:microbiology; D001232:Aspergillus fumigatus / Q000235:genetics / Q000302:isolation & purification*; D002176:Candida albicans / Q000235:genetics / Q000302:isolation & purification*; D002177:Candidiasis / Q000175:diagnosis* / Q000382:microbiology; D004271:DNA, Fungal / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D004271:DNA, Fungal