Article Figures & Data
Figures
- 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. fumigatusconidia (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.
