Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water Samples
<i>Legionella</i> are opportunistic intracellular pathogens that are found throughout the environment. The <i>Legionella</i> contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legio...
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| Format: | Article |
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MDPI AG
2022-07-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/10/7/1409 |
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| author | Montserrat Nácher-Vázquez Ana Barbosa Inês Armelim Andreia Sofia Azevedo Gonçalo Nieto Almeida Cristina Pizarro Nuno Filipe Azevedo Carina Almeida Laura Cerqueira |
| author_facet | Montserrat Nácher-Vázquez Ana Barbosa Inês Armelim Andreia Sofia Azevedo Gonçalo Nieto Almeida Cristina Pizarro Nuno Filipe Azevedo Carina Almeida Laura Cerqueira |
| author_sort | Montserrat Nácher-Vázquez |
| collection | DOAJ |
| description | <i>Legionella</i> are opportunistic intracellular pathogens that are found throughout the environment. The <i>Legionella</i> contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires’ disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus <i>Legionella</i>. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents’ autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3–4 h) and accurate detection of <i>Legionella</i>. |
| first_indexed | 2024-03-09T13:20:44Z |
| format | Article |
| id | doaj.art-bfbb4b8799824da5911f09bb3b5293e0 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-09T13:20:44Z |
| publishDate | 2022-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-bfbb4b8799824da5911f09bb3b5293e02023-11-30T21:30:48ZengMDPI AGMicroorganisms2076-26072022-07-01107140910.3390/microorganisms10071409Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water SamplesMontserrat Nácher-Vázquez0Ana Barbosa1Inês Armelim2Andreia Sofia Azevedo3Gonçalo Nieto Almeida4Cristina Pizarro5Nuno Filipe Azevedo6Carina Almeida7Laura Cerqueira8LEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, PortugalLEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, PortugalLEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, PortugalLEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, PortugalINIAV, IP—National Institute for Agrarian and Veterinary Research, Vairão, 4485-655 Vila Do Conde, PortugalINSA—National Institute of Health Doutor Ricardo Jorge, Rua Alexandre Herculano 321, 4000-055 Porto, PortugalLEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, PortugalLEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, PortugalLEPABE—Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal<i>Legionella</i> are opportunistic intracellular pathogens that are found throughout the environment. The <i>Legionella</i> contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires’ disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus <i>Legionella</i>. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents’ autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3–4 h) and accurate detection of <i>Legionella</i>.https://www.mdpi.com/2076-2607/10/7/1409fluorescence in situ hybridization<i>Legionella</i> sp.peptide nucleic acid (PNA)16S rRNA genewaterborne detection |
| spellingShingle | Montserrat Nácher-Vázquez Ana Barbosa Inês Armelim Andreia Sofia Azevedo Gonçalo Nieto Almeida Cristina Pizarro Nuno Filipe Azevedo Carina Almeida Laura Cerqueira Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water Samples Microorganisms fluorescence in situ hybridization <i>Legionella</i> sp. peptide nucleic acid (PNA) 16S rRNA gene waterborne detection |
| title | Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water Samples |
| title_full | Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water Samples |
| title_fullStr | Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water Samples |
| title_full_unstemmed | Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water Samples |
| title_short | Development of a Novel Peptide Nucleic Acid Probe for the Detection of <i>Legionella</i> spp. in Water Samples |
| title_sort | development of a novel peptide nucleic acid probe for the detection of i legionella i spp in water samples |
| topic | fluorescence in situ hybridization <i>Legionella</i> sp. peptide nucleic acid (PNA) 16S rRNA gene waterborne detection |
| url | https://www.mdpi.com/2076-2607/10/7/1409 |
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