Molecular beacons are sensitive fluorescent probes hybridizing selectively to designated DNA and RNA targets. They have recently become practical tools for quantitative real-time monitoring of single-stranded nucleic acids. Here, we comparatively study the performance of a variety of such probes, stemless and stem-containing DNA and PNA (peptide nucleic acid) beacons, in Tris-buffer solutions containing various concentrations of NaCl and MgCl(2). We demonstrate that different molecular beacons respond differently to the change of salt concentration, which could be attributed to the differences in their backbones and constructions. We have found that the stemless PNA beacon hybridizes rapidly to the complementary oligodeoxynucleotide and is less sensitive than the DNA beacons to the change of salt thus allowing effective detection of nucleic acid targets under various conditions. Though we found stemless DNA beacons improper for diagnostic purposes due to high background fluorescence, we believe that use of these DNA and similar RNA constructs in molecular-biophysical studies may be helpful for analysis of conformational flexibility of single-stranded nucleic acids. With the aid of PNA "openers", molecular beacons were employed for the detection of a chosen target sequence directly in double-stranded DNA (dsDNA). Conditions are found where the stemless PNA beacon strongly discriminates the complementary versus mismatched dsDNA targets. Together with the insensitivity of PNA beacons to the presence of salt and DNA-binding/processing proteins, the latter results demonstrate the potential of these probes as robust tools for recognition of specific sequences within dsDNA without denaturation and deproteinization of duplex DNA.

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A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluoresceinlabeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy.

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55°C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n ‫؍‬ 72), C. dubliniensis (n ‫؍‬ 58), C. glabrata (n ‫؍‬ 5), C. krusei (n ‫؍‬ 2), C. parapsilosis (n ‫؍‬ 4), and C. tropicalis (n ‫؍‬ 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.Brettanomyces is a well-recognized wine spoilage yeast that causes an undesirable flavor. The sensory character of this "Bretty" flavor is often described as mousiness, barnyard, horse sweat, or Band-Aid (5, 9). Current methods for identification and enumeration of Brettanomyces contamination take 1 to 2 weeks and rely on growth on a semiselective culture medium, followed by final identification by biochemical and physiological analysis and morphology as determined by microscopic examination (3). Morphological characterization of Brettanomyces is somewhat subjective, and there have been various morphological descriptions, such as bud scars, bullet shape, and Mickey Mouse-like. Newer techniques for rapid detection and identification of Brettanomyces, such as an enzyme-linked immunosorbent assay (7) and, more recently, PCR (6), have also been described.The nomenclature of Brettanomyces used in the wine industry differs from that of the recently revised taxonomy of yeasts (11,12). Enologists refer to the spoilage organism as Brettanomyces or "Brett" or, in some publications, by the species names Dekkera intermedia and Brettanomyces intermedius (3), Brettanomyces lambicus (3), Brettanomyces custersii, or Dekkera bruxellensis (6). Today, only D. bruxellensis is an accepted species name, and the other names are considered synonyms.Peptide nucleic acid (PNA) molecules are pseudopeptides which are able to hybridize to complementary nucleic acid targets (RNA and DNA) obeying Watson-Crick base pairing rules (2, 10). Due to their uncharged, neutral backbone, PNA probes exhibit favorable hybridization characteristics, such as high specificity, strong affinity, and rapid kinetics resulting in improved hybridization to highly structured targets, such as rRNA (13). In addition, the relatively hydrophobic character of PNAs compared to DNA oligonucleotides makes PNA probes capable of penetrating the hydrophobic cell wall following mild fixation conditions that do not lead to disruption of cell morphology (14). These unique characteristics of PNA have opened new possibilities for molecular diagnostic assays.The D1-D2 region of 26S ribosomal DNA (rDNA) of eucaryotic organisms shows a high degree of species variation and has been used for identification and taxonomy of yeast species (1,8). In this study, 26S rDNA sequence information was used to design speci...