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 aci ds in glass capillaries with melting curve analysis based on fluorescence r esonance energy transfer for the sensitive detection of point mutations in various settings, such as drug resistance and hereditary diseases. Point mu tations leading to an altered structure of lanosteroldemethylase, the targe t 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 fluconazol e-resistant C. tropicalis strains (minimum inhibitory concentration >128 mg /L), isolated from patients with AIDS, for the presence of defined point mu tations by comparing conventional cycle sequencing with a newly designed Li ghtCycler-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 a t position 1554 (T1554C). A heterozygous genotype was detected in 8 of 21 i solates by the LightCycler, but in only 3 of 21 isolates by conventional cy cle sequencing. In 2 of 13 C. albicans isolates, a homozygous point mutatio n 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. (C ) 2000 American Association for Clinical Chemistry.