J. Loeffler et al., Quantification of fungal DNA by using fluorescence resonance energy transfer and the Light Cycler system, J CLIN MICR, 38(2), 2000, pp. 586-590
The Light Cycler technique combines rapid in vitro amplification of DNA in
glass capillaries with real-time species determination and quantification o
f DNA load. We have established a quantitative PCR protocol for two clinica
lly important pathogens, Candida albicans and Aspergillus fumigatus. The se
nsitivity of the assay was comparable to those of previously described PCR
protocols (5 CFU/ml). Specific detection of C. albicans and A. fumigatus co
uld 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 quant
ification of the fungal loads in a limited number of clinical specimens fro
m patients with hematological malignancies and histologically proven invasi
ve fungal infections. Five of nine positive samples had fungal loads betwee
n 5 and 10 CFU/ml of blood, two of nine positive samples had fungal loads b
etween 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 po
sitive by PCR-enzyme-linked immunosorbent assay analysis.