Alexandra Bogožalec Košir (Author), Dane Lužnik (Author), Viktorija Tomič (Author), Mojca Milavec (Author)

Abstract

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.

Keywords

nukleinske kisline;dPCR;metode ekstrakcije DNK;gram-negativne bakterije;nucleic acid;DNA extraction methods;Gram-negative bacteria;

Data

Language: English
Year of publishing:
Typology: 1.01 - Original Scientific Article
Organization: NIB - National Institute of Biology
UDC: 60
COBISS: 149291011 Link will open in a new window
ISSN: 2079-6374
Views: 215
Downloads: 199
Average score: 0 (0 votes)
Metadata: JSON JSON-RDF JSON-LD TURTLE N-TRIPLES XML RDFA MICRODATA DC-XML DC-RDF RDF

Other data

Secondary language: Slovenian
Secondary keywords: nukleinske kisline;dPCR;metode ekstrakcije DNK;gram-negativne bakterije;
Source comment: Soavtorji: Dane Lužnik, Viktorija Tomič and Mojca Milavec; Nasl. z nasl. zaslona; Opis vira z dne 17. 4. 2023;
Pages: str. [1]-15
Volume: ǂiss. ǂ4
Issue: ǂart. ǂ463
Chronology: Apr. 2023
DOI: 10.3390/bios13040463
ID: 24512635