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Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of legionellae in biofilms

Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of legionellae in biofilms
Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of legionellae in biofilms
Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.
0099-2240
5453-5462
Wilks, S.A.
86c1f41a-12b3-451c-9245-b1a21775e993
Keevil, C.W.
cb7de0a7-ce33-4cfa-af52-07f99e5650eb
Wilks, S.A.
86c1f41a-12b3-451c-9245-b1a21775e993
Keevil, C.W.
cb7de0a7-ce33-4cfa-af52-07f99e5650eb

Wilks, S.A. and Keevil, C.W. (2006) Targeting species-specific low-affinity 16S rRNA binding sites by using peptide nucleic acids for detection of legionellae in biofilms. Applied and Environmental Microbiology, 72 (8), 5453-5462. (doi:10.1128/AEM.02918-05).

Record type: Article

Abstract

Using fluorescence in situ hybridization to detect bacterial groups has several inherent limitations. DNA probes are generally used, targeting sites on the 16S rRNA. However, much of the 16S rRNA is highly conserved, with variable regions often located in inaccessible areas where secondary structures can restrict probe access. Here, we describe the use of peptide nucleic acid (PNA) probes as a superior alternative to DNA probes, especially when used for environmental samples. A complex bacterial genus (Legionella) was studied, and two probes were designed, one to detect all species and one targeted to Legionella pneumophila. These probes were developed from existing sequences and are targeted to low-binding-affinity sites on the 16S rRNA. In total, 47 strains of Legionella were tested. In all cases, the Legionella spp. PNA probe labeled cells strongly but did not bind to any non-Legionella species. Likewise, the specific L. pneumophila PNA probe labeled only strains of L. pneumophila. By contrast, the equivalent DNA probes performed poorly. To assess the applicability of this method for use on environmental samples, drinking-water biofilms were spiked with a known concentration of L. pneumophila bacteria. Quantifications of the L. pneumophila bacteria were compared using PNA hybridization and standard culture methods. The culture method quantified only 10% of the number of L. pneumophila bacteria found by PNA hybridization. This illustrates the value of this method for use on complex environmental samples, especially where cells may be in a viable but noncultivable state.

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More information

Submitted date: 12 December 2005
Published date: 1 August 2006

Identifiers

Local EPrints ID: 56506
URI: http://eprints.soton.ac.uk/id/eprint/56506
ISSN: 0099-2240
PURE UUID: acbcc2ce-06fb-43a1-b967-79559c0fb512
ORCID for S.A. Wilks: ORCID iD orcid.org/0000-0002-4134-9415
ORCID for C.W. Keevil: ORCID iD orcid.org/0000-0003-1917-7706

Catalogue record

Date deposited: 06 Aug 2008
Last modified: 16 Mar 2024 03:24

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