Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp.
The <i>Achromobacter</i> 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 bet...
Saved in:
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2025-03-01
|
| Series: | Pathogens |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-0817/14/3/271 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850090911455772672 |
|---|---|
| author | Giulia Maria Saitta Laura Veschetti Rebecca Feletti Angela Sandri Marzia Boaretti Paola Melotti Maria Carelli Maria M. Lleò Giovanni Malerba Caterina Signoretto |
| author_facet | Giulia Maria Saitta Laura Veschetti Rebecca Feletti Angela Sandri Marzia Boaretti Paola Melotti Maria Carelli Maria M. Lleò Giovanni Malerba Caterina Signoretto |
| author_sort | Giulia Maria Saitta |
| collection | DOAJ |
| description | The <i>Achromobacter</i> 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 <i>Achromobacter</i> 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: <i>A. xylosoxidans</i>, <i>A. dolens</i>, and <i>A. insuavis</i>. Two additional probes were designed for novel genotypes unrelated to publicly available sequences. The LOD ranged from 0.005 pg/µL to 1 pg/µL. 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. |
| format | Article |
| id | doaj-art-8f9ad157e79540cd99351f69e71298bc |
| institution | DOAJ |
| issn | 2076-0817 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Pathogens |
| spelling | doaj-art-8f9ad157e79540cd99351f69e71298bc2025-08-20T02:42:28ZengMDPI AGPathogens2076-08172025-03-0114327110.3390/pathogens14030271Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp.Giulia Maria Saitta0Laura Veschetti1Rebecca Feletti2Angela Sandri3Marzia Boaretti4Paola Melotti5Maria Carelli6Maria M. Lleò7Giovanni Malerba8Caterina Signoretto9Diagnostic and Public Health Department, University of Verona, 37134 Verona, ItalyInfections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, 20132 Milano, ItalyDiagnostic and Public Health Department, University of Verona, 37134 Verona, ItalyDiagnostic and Public Health Department, University of Verona, 37134 Verona, ItalyDiagnostic and Public Health Department, University of Verona, 37134 Verona, ItalyCystic Fibrosis Centre, Azienda Ospedaliera Universitaria Integrata Verona, Piazzale A. Stefani 1, 37126 Verona, ItalyCardiology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, 40138 Bologna, ItalyDiagnostic and Public Health Department, University of Verona, 37134 Verona, ItalyGMLab, Department of Surgical Sciences, Dentistry, Gynaecology and Paediatrics, University of Verona, 37134 Verona, ItalyDiagnostic and Public Health Department, University of Verona, 37134 Verona, ItalyThe <i>Achromobacter</i> 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 <i>Achromobacter</i> 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: <i>A. xylosoxidans</i>, <i>A. dolens</i>, and <i>A. insuavis</i>. Two additional probes were designed for novel genotypes unrelated to publicly available sequences. The LOD ranged from 0.005 pg/µL to 1 pg/µL. 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.https://www.mdpi.com/2076-0817/14/3/271<i>Achromobacter</i>identificationcystic fibrosisqPCR |
| spellingShingle | Giulia Maria Saitta Laura Veschetti Rebecca Feletti Angela Sandri Marzia Boaretti Paola Melotti Maria Carelli Maria M. Lleò Giovanni Malerba Caterina Signoretto Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp. Pathogens <i>Achromobacter</i> identification cystic fibrosis qPCR |
| title | Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp. |
| title_full | Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp. |
| title_fullStr | Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp. |
| title_full_unstemmed | Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp. |
| title_short | Development of a Simple and Accurate Molecular Protocol Using 16SrRNA for Species-Specific Identification of <i>Achromobacter</i> spp. |
| title_sort | development of a simple and accurate molecular protocol using 16srrna for species specific identification of i achromobacter i spp |
| topic | <i>Achromobacter</i> identification cystic fibrosis qPCR |
| url | https://www.mdpi.com/2076-0817/14/3/271 |
| work_keys_str_mv | AT giuliamariasaitta developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT lauraveschetti developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT rebeccafeletti developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT angelasandri developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT marziaboaretti developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT paolamelotti developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT mariacarelli developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT mariamlleo developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT giovannimalerba developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp AT caterinasignoretto developmentofasimpleandaccuratemolecularprotocolusing16srrnaforspeciesspecificidentificationofiachromobacterispp |