Purpose: To develop new PNA-FISH probes specific for C. albicans and compare their performance against DNA probes targeting the same regions on the 28S rRNA; to use flow cytometry (FCM) for quantitative analysis of probe hybridization.
Methods: Two sets of DNA probes and their corresponding PNA counterparts were selected from alignments of the 28S rDNA of representative Candida species. One target region was recently shown by our group to be addressable with the DNA-FISH probe Calb2208. The other probe region is unique and has not been previously described. Fluorescently-labeled DNA and PNA probes having similar melting temperatures were evaluated against a panel of yeasts, including C. albicans and other Candida spp. Fluorescence microscopy and benchtop FCM (BD Accuri C6) were used for qualitative and quantitative analyses, respectively. Probes were evaluated for hybridization intensity, staining uniformity and specificity for C. albicans.
Results: Overall, the PNA probes were brighter and provided more uniform staining than the DNA probes. PNA CalB2208 was brighter than PNA 28S rRNA-3, but also yielded higher non-specific staining. Ratiometric analysis of FCM data indicated that PNA 28S rRNA-3 was 2.5 – 5-fold more discriminatory than PNA CalB2208.
Significance: PNA-FISH was shown to be diagnostically superior to DNA-FISH in this application. A novel probe, 28S rRNA-3, was developed and shown to be highly discriminatory for C. albicans. This probe may enable rapid, selective identification of C. albicans in both foods and in clinical samples.