Improved DNA-FISH for cytometric detection ofCandidaspp

Abstract

Aims: We developed improved methods for DNA‐based fluorescence in situ hybridization (FISH) for rapid detection of Candida spp. and Candida albicans via flow cytometry. Methods and Results: Two previously reported C. albicans‐targeted DNA probes were evaluated against whole cells of C. albicans and related Candida species using a rapid, high‐temperature hybridization protocol. One probe (CalB2208) was shown for the first time to be suitable as a FISH probe. Although cell labelling for both probes was relatively bright, we were able to substantially improve our results by altering fixation and hybridization conditions. For fixation, a 60 : 40 mixture of 10% buffered formalin and ethanol was most effective. Probe intensity was improved as much as ten‐fold through the use of unlabelled helper probes, and buffer containing 0·9 mol l−1 NaCl plus 10% formamide yielded the best hybridizations for both probe/helper cocktails. Although optimal labelling occurred with longer hybridizations, we found that C. albicans could be completely differentiated from the nontarget yeast Rhodotorula glutinis after only 15 min using the brightest probe (Calb‐1249) and that a formal washing step was not required. Specificities of probe/helper cocktails under optimal conditions were determined using a panel of target and nontarget cell types, including four strains of Candida dubliniensis. Calb‐1249 cross‐reacted slightly with Candida parapsilosis and strongly with both Candida tropicalis and C. dubliniensis. In contrast, we found that CalB2208 was exclusive for C. albicans. The molecular basis of this specificity was confirmed by DNA sequencing. Conclusions: We describe DNA probe‐based approaches for rapid and bright labelling of Candida spp. and for specific labelling of C. albicans without cross‐reaction with C. dubliniensis. Our work improves upon previously described methods. Significance and Impact of the Study: The methods described here for rapid FISH‐based detection of Candida spp. may have applications in both clinical and food microbiology.

Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

“…Although the results for D-CalB2208 were in agreement with data from our previous report (23), D-Ca720 reacted strongly with C. tropicalis ATCC 750 (data not shown). This can be explained by fewer mismatches in the C. tropicalis sequence and contiguous matches with this probe on both ends of the target sequence (Fig.…”

“…The original motivation for this study was to examine the impact of the use of PNA technology on the quality of hybridizations with CalB2208, which we had demonstrated in a previous report to be both suitable for DNA-FISH and specific for C. albicans (23). Although we had achieved satisfactory results with DNA-FISH, we hypothesized that the use of PNA would result in the brighter, faster, and more uniform hybridizations typical of PNA chemistry (25).…”

“…Ideally, cells whose ribosomes have been targeted with FISH probes are brightly labeled and are easily differentiated from nontarget cells. A variety of groups have reported the use of DNA-based FISH for the identification of C. albicans using probes targeting the 18S rRNA subunit; however, either the specificities of some of these probes have not been rigorously examined or the probes have been subsequently shown to react with nontarget yeasts (11)(12)(13)(14)(15)23). More recently, probes based on alternative chemistries, such as peptide nucleic acid (PNA), have been used for the identification of microbial pathogens.…”

“…Once the optimal conditions for FISH with P-CalB2208 and P-Ca726 were determined, we performed a direct comparison between these probes and their DNA counterparts, using the DNA hybridization conditions reported in our previous work (23). DNA probes were examined with and without helpers.…”

“…The present study builds on our previous work using DNAbased FISH (DNA-FISH) for detection of C. albicans, with a key interest being whether further improvements in performance characteristics could be gained through the use of PNA technology. In our previous work, we evaluated CalB2208, a 28S rRNAtargeted DNA probe reported in the patent literature, and found it to be both suitable for FISH and specific for C. albicans (23). The purpose of this study was 3-fold: (i) to evaluate the performance of a PNA version of CalB2208 and to compare its performance to that of its DNA counterpart, (ii) to screen for and identify new regions within the 28S rRNA gene that may allow specific detection of C. albicans or other clinically important Candida spp., and (iii) to evaluate the performance of C. albicans-targeted PNA probes resulting from this screen by using both qualitative (fluorescence microscopy [FM]) and quantitative (flow cytometry [FCM]) methods.…”