Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system.
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.
Loeffler
J
J
Medizinische Klinik, Abteilung II, Eberhard-Karls-Universität, 72076 Tübingen, Germany. juergen.loeffler@med.uni-tuebingen.de
Henke
N
N
Hebart
H
H
Schmidt
D
D
Hagmeyer
L
L
Schumacher
U
U
Einsele
H
H
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
D004271:DNA, Fungal