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.

FIG. 1

Hybridization probe format overview. Pr. 1 and Pr. 2, primers amplifying a conserved region of the 18S rRNA gene. Probe FL is labeled with fluorescein, and probe LC is labeled with Light Cycler Red 640 fluorophore. During annealing, the excitation energy is transferred to the acceptor fluorophore, Light Cycler Red 640 fluorophore. The emitted fluorescence is proportional to the amount of DNA generated during the PCR. After the completion of polymerization, the emission of fluorescence is stopped.

FIG. 2

Quantification of serially diluted A. fumigatus conidia (10⁴ to 10¹ CFU) by using the Light Cycler-based PCR technique (A) and Light Cycler-based standard curve report for serially diluted A. fumigatus conidia (10⁴ to 10¹ CFU) (B).

FIG. 3

Agarose gel electrophoresis of serially diluted A. fumigatus conidia (10⁶ to 10⁰ CFU, corresponding to 10 ng to 10 fg of DNA) showing a single, specific band at 500 bp. DNA was extracted as described in the text and was amplified with a conventional thermocycler (lane 2, positive control) and by the Light Cycler™ technique (lanes 3 to 9). Lanes: 1, 100-bp ladder; 2, 10⁶ CFU (conventional thermocycler); 3, 10⁵ CFU (1 ng); 4, 10⁴ CFU (100 pg); 5, 10³ CFU (10 pg); 6, 10² CFU (1 pg); 7, 10¹ CFU (100 fg); 8, 10⁰ CFU (10 fg); 9, negative control (double-distilled H₂O).

FIG. 4

Sensitivity and reproducibility of the Light Cycler-based detection of fungal DNA. Bars: 1, 5 CFU (sensitivity control); 2, negative control (double-distilled H₂O); 3 to 5, 10¹ CFU (identical DNA extraction); 6 to 8, 10² CFU (identical DNA extraction); 9 to 11, 10³ CFU (identical DNA extraction); 12 to 14, 10⁴ CFU (identical DNA extraction); 15, negative control (double-distilled H₂O); 16 to 18, 10¹ CFU (different dilution series); 19 to 21, 10² CFU (different dilution series); 22 to 24, 10³ CFU (different dilution series); 25, negative control (double-distilled H₂O); 26 to 34, patient samples.