title: Identification of indicator microorganisms using a standardized PNA FISH method.
abstract: A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings. The S. aureus and P. aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E. coli PNA probe, as expected from sequence data, also detected Shigella species. The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology.
authors.affiliation: Boston Probes, Bedford, MA, USA. hokeefe@bostonprobes.com
authors.forename: H
authors.initials: H
authors.lastname: Perry-O'Keefe
authors.forename: S
authors.initials: S
authors.lastname: Rigby
authors.forename: K
authors.initials: K
authors.lastname: Oliveira
authors.forename: D
authors.initials: D
authors.lastname: Sørensen
authors.forename: H
authors.initials: H
authors.lastname: Stender
authors.forename: J
authors.initials: J
authors.lastname: Coull
authors.forename: J J
authors.initials: JJ
authors.lastname: Hyldig-Nielsen
mesh_terms: D006090:Gram-Negative Bacteria / Q000235:genetics* / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000235:genetics* / Q000302:isolation & purification; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008828:Microbiological Techniques; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D012335:RNA, Ribosomal / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity; D015003:Yeasts / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D012335:RNA, Ribosomal