| Title: | Evaluation of DNA extraction methods for reliable quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa |
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| Authors: | ID Bogožalec Košir, Alexandra (Author)
ID Lužnik, Dane (Author)
ID Tomič, Viktorija (Author)
ID Milavec, Mojca (Author) |
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| Files: |  URL - Source URL, visit https://doi.org/10.3390/bios13040463
 PDF - Presentation file, download (1,59 MB)
MD5: C89877D282091DB80CD175C2BA236EE0
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| Language: | English |
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| Typology: | 1.01 - Original Scientific Article |
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| Organization: |  NIB - National Institute of Biology
 UKPBAG - University Clinic of Respiratory and Allergic Diseases Golnik
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| 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. |
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| Keywords: | nucleic acid, dPCR, DNA extraction methods, Gram-negative bacteria |
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| Publication status: | Published |
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| Publication version: | Version of Record |
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| Publication date: | 06.04.2023 |
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| Year of publishing: | 2023 |
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| Number of pages: | str. 2458-2469 |
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| Numbering: | iss. 9, Vol. 79 |
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| PID: | 20.500.12556/DiRROS-19255  |
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| UDC: | 60 |
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| ISSN on article: | 2079-6374 |
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| DOI: | 10.3390/bios13040463 |
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| COBISS.SI-ID: | 149291011 |
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| Note: | Soavtorji: Dane Lužnik, Viktorija Tomič and Mojca Milavec;
Nasl. z nasl. zaslona;
Opis vira z dne 17. 4. 2023;
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| Publication date in DiRROS: | 12.07.2024 |
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| Views: | 357 |
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| Downloads: | 277 |
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