20.500.12556/DiRROS-20758 Development of novel digital PCR assays for the rapid quantification of Gram-negative bacteria biomarkers using RUCS algorithm 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. digital PCR (dPCR) Gram-negative bacteria pathogen detection respiratory infections biomarkers RUCS algorithm digitalna PCR Gram-negativne bakterije detekcija patogenov okužba dihal biomarkerji diagnostika molekularna biologija true false true Angleški jezik Slovenski jezik Neznano 2024-11-05 09:51:03 2024-11-05 09:51:03 2024-11-29 03:38:13 0000-00-00 00:00:00 2024 0 0 Soavtorji: Špela Alič, Viktorija Tomič, Dane Lužnik, Tanja Dreo, Mojca Milavec; str. 72-80 Vol. 232 Dec. 2024 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2024-12-01 579 1046-2023 10.1016/j.ymeth.2024.10.011 213682691 https://doi.org/10.1016/j.ymeth.2024.10.011 1 1920d6b1-9b53-11ef-89f3-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=29293 RAZ_Bogozalec_Kosir_Alexandra_2024.pdf RAZ_Bogozalec_Kosir_Alexandra_2024.pdf 1 91FD8FB54941BE1D49655A318AED9464 7e6e587acda7a47549a5900ed0ca46eb47a7b5ec97a00ff068e8fcdda1fff5bf 5f4071cf-ad69-11ef-ba3f-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=29541 Nacionalni inštitut za biologijo 0 0 0