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Query: "keywords" (DNA extraction) .

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1.
Evaluation of DNA extraction methods for reliable quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa
Alexandra Bogožalec Košir, Dane Lužnik, Viktorija Tomič, Mojca Milavec, 2023, original scientific article

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: nucleic acid, dPCR, DNA extraction methods, Gram-negative bacteria
Published in DiRROS: 12.07.2024; Views: 55; Downloads: 20
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2.
The influence of storage conditions and DNA extraction protocol on the results of molecular analysis of the European spruce bark beetle (Ips typographus L.)
Zina Devetak, Andreja Kavčič, Maarten De Groot, Barbara Piškur, 2023, original scientific article

Abstract: One of the key steps of the molecular identification of bark beetles is obtaining a sufficient quantity of high-quality DNA extract. In this study, we investigated the influence of different storage procedures for Ips typographus (L.) specimens and various DNA extraction protocols on the quantity and quality of DNA intended for use in molecular diagnostics. Adult beetles were frozen at -20 °C, either dry or in ethanol. We tested four different protocols for DNA extraction. We compared the quantity of extracted DNA and assessed its quality with PCR and Sanger sequencing. Different storage protocols had no significant effect on the quantity of DNA extracted. However, freezing specimens in ethanol provided higher-quality DNA for molecular applications. Only two of the extraction protocols produced sequenceable amplicons, and the difference in the amount of extracted DNA between them was not significant. We propose the optimal combination of storing specimens in ethanol at -20°C and using the Nucleospin Insect DNA extraction kit from Macherey Nagel, enabling a timeefficient identification process.
Keywords: early detection, specimen storage, total DNA extraction, PCR, polymerase chain reaction, Sanger sequencing, molecular diagnostics
Published in DiRROS: 02.02.2024; Views: 714; Downloads: 257
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