Rapid Identification and Differentiation of Candida albicans and Candida dubliniensis by Capillary-Based Amplification and Fluorescent Probe Hybridization

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Abstract
We developed a rapid genotypic assay to differentiate the germ tube-positive yeasts Candida albicans and Candida dubliniensis . Fluorescently labeled nucleic acid probe binding and subsequent denaturation from the target site in the PCR amplicons produced characteristic peak melting temperatures ( T m ) that identified each species. Peak T m s of C. albicans ( n = 69) and C. dubliniensis ( n = 28) isolates produced in the presence of their respective probes were 61.04 ± 0.64°C and 60.52 ± 1.01°C (averages ± standard deviations). No signal was generated when the C. albicans or C. dubliniensis probes were tested against DNA from their counterparts. Both probes reacted with Candida tropicalis DNA, but the T m was 51.85 ± 0.05°C with the C. albicans probe and 51.92 ± 0.10°C with the C. dubliniensis probe, differentiating C. tropicalis DNA from C. albicans and C. dubliniensis. A novel hybrid probe was designed to identify both species in a single reaction based on a 4°C difference in peak T m s. Our assay is rapid (≤2 h) and allows reliable detection and differentiation of the two germ tube-positive Candida spp.