Rapid detection of point mutations by fluorescence resonance energy transfer and probe melting curves in Candida species.
BACKGROUND
The LightCycler(TM) combines rapid amplification of nucleic acids in glass capillaries with melting curve analysis based on fluorescence resonance energy transfer for the sensitive detection of point mutations in various settings, such as drug resistance and hereditary diseases. Point mutations leading to an altered structure of lanosteroldemethylase, the target enzyme of the fungistatic azoles, are an important mechanism of acquired resistance in Candida albicans.


METHODS
We screened 13 fluconazole-resistant C. albicans and 21 fluconazole-resistant C. tropicalis strains (minimum inhibitory concentration >128 mg/L), isolated from patients with AIDS, for the presence of defined point mutations by comparing conventional cycle sequencing with a newly designed LightCycler-based assay.


RESULTS
In C. tropicalis, 5 of 21 isolates showed the wild-type sequence, and 8 of 21 showed the homozygous nucleotide exchange thymine to cytosine at position 1554 (T1554C). A heterozygous genotype was detected in 8 of 21 isolates by the LightCycler, but in only 3 of 21 isolates by conventional cycle sequencing. In 2 of 13 C. albicans isolates, a homozygous point mutation leading to an amino acid exchange at position 464 (glycine to serine) was detected in both assays.


CONCLUSIONS
The LightCycler technique offers standardized, fast, sensitive, and reproducible detection of point mutations in different Candida spp.
Loeffler
J
J
Medizinische Klinik, Abteilung II, Otfried-Mueller-Strasse 10, 72076 Tuebingen, Germany. Fax 49-7071-293179, USA. juergen.loeffler@med.uni-tuebingen.de
Hagmeyer
L
L
Hebart
H
H
Henke
N
N
Schumacher
U
U
Einsele
H
H
D000163:Acquired Immunodeficiency Syndrome / Q000382:microbiology; D000935:Antifungal Agents / Q000494:pharmacology; D002175:Candida / Q000235:genetics* / Q000302:isolation & purification; D004271:DNA, Fungal / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D015725:Fluconazole / Q000494:pharmacology; D006801:Humans; D008826:Microbial Sensitivity Tests; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D000935:Antifungal Agents; D004271:DNA, Fungal; D015725:Fluconazole