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The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires\u2019 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 Legionella. 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\u2019 autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3\u20134 h) and accurate detection of Legionella.<\/jats:p>","DOI":"10.3390\/microorganisms10071409","type":"journal-article","created":{"date-parts":[[2022,7,14]],"date-time":"2022-07-14T00:12:40Z","timestamp":1657757560000},"page":"1409","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Development of a Novel Peptide Nucleic Acid Probe for the Detection of Legionella spp. in Water Samples"],"prefix":"10.3390","volume":"10","author":[{"given":"Montserrat","family":"N\u00e1cher-V\u00e1zquez","sequence":"first","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"INIAV, IP\u2014National Institute for Agrarian and Veterinary Research, Vair\u00e3o, 4485-655 Vila Do Conde, Portugal"}]},{"given":"Ana","family":"Barbosa","sequence":"additional","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"ALiCE\u2014Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"given":"In\u00eas","family":"Armelim","sequence":"additional","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0521-2841","authenticated-orcid":false,"given":"Andreia Sofia","family":"Azevedo","sequence":"additional","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"ALiCE\u2014Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"i3S\u2014Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, 4200-135 Porto, Portugal"},{"name":"IPATIMUP\u2014Instituto de Patologia e Imunologia Molecular, Universidade do Porto, 4200-135 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1513-0951","authenticated-orcid":false,"given":"Gon\u00e7alo Nieto","family":"Almeida","sequence":"additional","affiliation":[{"name":"INIAV, IP\u2014National Institute for Agrarian and Veterinary Research, Vair\u00e3o, 4485-655 Vila Do Conde, Portugal"}]},{"given":"Cristina","family":"Pizarro","sequence":"additional","affiliation":[{"name":"INSA\u2014National Institute of Health Doutor Ricardo Jorge, Rua Alexandre Herculano 321, 4000-055 Porto, Portugal"}]},{"given":"Nuno Filipe","family":"Azevedo","sequence":"additional","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"ALiCE\u2014Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0175-1119","authenticated-orcid":false,"given":"Carina","family":"Almeida","sequence":"additional","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"INIAV, IP\u2014National Institute for Agrarian and Veterinary Research, Vair\u00e3o, 4485-655 Vila Do Conde, Portugal"},{"name":"Centre of Biological Engineering (CEB), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8452-4749","authenticated-orcid":false,"given":"Laura","family":"Cerqueira","sequence":"additional","affiliation":[{"name":"LEPABE\u2014Laboratory for Process Engineering, Environment, Biotechnology and Energy, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"},{"name":"ALiCE\u2014Associate Laboratory in Chemical Engineering, Faculty of Engineering, University of Porto, Rua Dr. Roberto Frias, 4200-465 Porto, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,13]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1122","DOI":"10.1080\/21505594.2021.1903199","article-title":"Pathogenicity and Virulence of Legionella: Intracellular Replication and Host 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