20.500.12556/DiRROS-19255 Evaluation of DNA extraction methods for reliable quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used. nucleic acid dPCR DNA extraction methods Gram-negative bacteria nukleinske kisline dPCR metode ekstrakcije DNK gram-negativne bakterije true false true Angleški jezik Slovenski jezik Neznano 2024-07-12 03:38:04 2024-07-12 09:01:17 2024-09-12 10:07:47 0000-00-00 00:00:00 2023 0 0 Soavtorji: Dane Lužnik, Viktorija Tomič and Mojca Milavec; Nasl. z nasl. zaslona; Opis vira z dne 17. 4. 2023; str. 2458-2469 Vol. 79 iss. 9 Sept. 2023 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2023-04-06 60 2079-6374 10.3390/bios13040463 149291011 https://doi.org/10.3390/bios13040463 1 622b671d-3fef-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26515 RAZ_Bogozalec_Kosir_Alexandra_2023.pdf RAZ_Bogozalec_Kosir_Alexandra_2023.pdf 1 C89877D282091DB80CD175C2BA236EE0 3219708e7eb12b413f619c5e90399e9a642fdf07c6cb3e20a85369bcd176bb95 b5678466-401b-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26554 Nacionalni inštitut za biologijo Univerzitetna klinika za pljučne bolezni in alergijo Golnik 0 0 0