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Title:Development of novel digital PCR assays for the rapid quantification of Gram-negative bacteria biomarkers using RUCS algorithm
Authors:ID Bogožalec Košir, Alexandra (Author)
ID Alič, Špela (Author)
ID Tomič, Viktorija (Author)
ID Lužnik, Dane (Author)
ID Dreo, Tanja (Author)
ID Milavec, Mojca (Author)
Files:URL URL - Source URL, visit https://doi.org/10.1016/j.ymeth.2024.10.011
 
Language:English
Typology:1.01 - Original Scientific Article
Organization:Logo NIB - National Institute of Biology
Abstract:Rapid and accurate identification of bacterial pathogens is crucial for effective treatment and infection control, particularly in hospital settings. Conventional methods like culture techniques and MALDI-TOF mass spectrometry are often time-consuming and less sensitive. This study addresses the need for faster and more precise diagnostic methods by developing novel digital PCR (dPCR) assays for the rapid quantification of biomarkers from three Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Utilizing publicly available genomes and the rapid identification of PCR primers for unique core sequences or RUCS algorithm, we designed highly specific dPCR assays. These assays were validated using synthetic DNA, bacterial genomic DNA, and DNA extracted from clinical samples. The developed dPCR methods demonstrated wide linearity, a low limit of detection (approx. 30 copies per reaction), and robust analytical performance with measurement uncertainty below 25 %. The assays showed high repeatability and intermediate precision, with no cross-reactivity observed. Comparison with MALDI-TOF mass spectrometry revealed substantial concordance, highlighting the methods’ suitability for clinical diagnostics. This study underscores the potential of dPCR for rapid and precise quantification of Gram-negative bacterial biomarkers. The developed methods offer significant improvements over existing techniques, providing faster, more accurate, and SI-traceable measurements. These advancements could enhance clinical diagnostics and infection control practices.
Keywords:digital PCR (dPCR), Gram-negative bacteria, pathogen detection, respiratory infections, biomarkers, RUCS algorithm
Publication status:Published
Publication version:Version of Record
Publication date:01.12.2024
Year of publishing:2024
Number of pages:str. 72-80
Numbering:Vol. 232
PID:20.500.12556/DiRROS-20758 New window
UDC:579
ISSN on article:1046-2023
DOI:10.1016/j.ymeth.2024.10.011 New window
COBISS.SI-ID:213682691 New window
Note:Soavtorji: Špela Alič, Viktorija Tomič, Dane Lužnik, Tanja Dreo, Mojca Milavec;
Publication date in DiRROS:05.11.2024
Views:97
Downloads:23
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Record is a part of a journal

Title:Methods
Shortened title:Methods
Publisher:Academic Press
ISSN:1046-2023
COBISS.SI-ID:862740 New window

Document is financed by a project

Funder:ARIS - Slovenian Research and Innovation Agency
Project number:Z2-1860-2019
Name:Meroslovje za sledenje bolnišničnih okužb respiratornega trakta, ki jih povzročajo Gram-negativne bakterije

Funder:ARIS - Slovenian Research and Innovation Agency
Project number:P4-0165-2022
Name:Biotehnologija in sistemska biologija rastlin

Funder:EC - European Commission
Funding programme:SEPTIMET

Funder:Other - Other funder or multiple funders
Funding programme:European Union, European Regional Development Fund

Licences

License:CC BY 4.0, Creative Commons Attribution 4.0 International
Link:http://creativecommons.org/licenses/by/4.0/
Description:This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

Language:Slovenian
Keywords:digitalna PCR, Gram-negativne bakterije, detekcija patogenov, okužba dihal, biomarkerji, diagnostika, molekularna biologija


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