Rapid detection of point mutations by fluorescence resonance energy transfer and probe melting curves in Candida species.

Important note: Information in this article was accurate in 2000. The state of the art may have changed since the publication date.

Rapid detection of point mutations by fluorescence resonance energy transfer and probe melting curves in Candida species.

Clin Chem. 2000 May;46(5):631-5. Unique Identifier : AIDSLINE MED/20256813
Loeffler J; Hagmeyer L; Hebart H; Henke N; Schumacher U; Einsele H; Medizinische Klinik, Abteilung II, Otfried-Mueller-Strasse 10,; 72076 Tuebingen, Germany. Fax 49-7071-293179, USA.; juergen.loeffler@med.uni-tuebingen.de

Abstract: 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. CONCLUSION: The LightCycler technique offers standardized, fast, sensitive, and reproducible detection of point mutations in different Candida spp.

Keywords: JOURNAL ARTICLE Acquired Immunodeficiency Syndrome/MICROBIOLOGY Antifungal Agents/PHARMACOLOGY Candida/*GENETICS/ISOLATION & PURIF DNA, Fungal/GENETICS Electrophoresis, Agar Gel Energy Transfer Fluconazole/PHARMACOLOGY Fluorescence Human Microbial Sensitivity Tests *Point Mutation Polymerase Chain Reaction Reproducibility of Results Sensitivity and Specificity

000830
A0080983

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