{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T11:27:36Z","timestamp":1792063656998,"version":"4.4.0"},"reference-count":51,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T00:00:00Z","timestamp":1741737600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>The Achromobacter genus comprises 22 species and various genogroups. Some species with higher virulence or antibiotic resistance are more likely to cause chronic infections in people with cystic fibrosis (CF). Current identification methods often fail to accurately distinguish between the species or result in misidentifications due to biochemical similarities. This study aims to develop an accurate qPCR protocol for species-level identification that is applicable in clinical diagnostic laboratories. Whole-genome sequencing of clinical isolates from different Achromobacter species identified species-specific single-nucleotide polymorphisms (SNPs) in two 16S gene regions. Based on these SNPs, two sets of primers and qPCR probes were designed to generate unique identification profiles. Thermal profiles were optimized, and qPCR was performed on serial bacterial DNA dilutions to determine the detection limit (LOD). Four probes successfully identified three species: A. xylosoxidans, A. dolens, and A. insuavis. Two additional probes were designed for novel genotypes unrelated to publicly available sequences. The LOD ranged from 0.005 pg\/\u00b5L to 1 pg\/\u00b5L. Combined probes achieved 100% sensitivity, with specificity ranging from 97.95% to 100%. This qPCR protocol enables accurate species identification, overcoming the limitations of current methods, and represents a reliable tool for clinical diagnostics.<\/jats:p>","DOI":"10.3390\/pathogens14030271","type":"journal-article","created":{"date-parts":[[2025,3,12]],"date-time":"2025-03-12T10:45:06Z","timestamp":1741776306000},"page":"271","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of Achromobacter spp."],"prefix":"10.3390","volume":"14","author":[{"given":"Giulia Maria","family":"Saitta","sequence":"first","affiliation":[{"name":"Diagnostic and Public Health Department, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0413-6031","authenticated-orcid":false,"given":"Laura","family":"Veschetti","sequence":"additional","affiliation":[{"name":"Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milano, Italy"},{"name":"Vita-Salute San Raffaele University, 20132 Milano, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-6136-5668","authenticated-orcid":false,"given":"Rebecca","family":"Feletti","sequence":"additional","affiliation":[{"name":"Diagnostic and Public Health Department, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5107-0960","authenticated-orcid":false,"given":"Angela","family":"Sandri","sequence":"additional","affiliation":[{"name":"Diagnostic and Public Health Department, University of Verona, 37134 Verona, Italy"},{"name":"General and Upper GI Surgery Division, Azienda Ospedaliera Universitaria Integrata Verona, Piazzale A. Stefani 1, 37126 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marzia","family":"Boaretti","sequence":"additional","affiliation":[{"name":"Diagnostic and Public Health Department, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5276-1595","authenticated-orcid":false,"given":"Paola","family":"Melotti","sequence":"additional","affiliation":[{"name":"Cystic Fibrosis Centre, Azienda Ospedaliera Universitaria Integrata Verona, Piazzale A. Stefani 1, 37126 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5518-6226","authenticated-orcid":false,"given":"Maria","family":"Carelli","sequence":"additional","affiliation":[{"name":"Cardiology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, 40138 Bologna, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria M.","family":"Lle\u00f2","sequence":"additional","affiliation":[{"name":"Diagnostic and Public Health Department, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8705-8560","authenticated-orcid":false,"given":"Giovanni","family":"Malerba","sequence":"additional","affiliation":[{"name":"GMLab, Department of Surgical Sciences, Dentistry, Gynaecology and Paediatrics, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caterina","family":"Signoretto","sequence":"additional","affiliation":[{"name":"Diagnostic and Public Health Department, University of Verona, 37134 Verona, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,3,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2074","DOI":"10.1128\/JCM.02556-16","article-title":"Prevalence and outcomes of Achromobacter species infections in adults with cystic fibrosis: A North American cohort study","volume":"55","author":"Edwards","year":"2017","journal-title":"J. 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