20.500.12556/DiRROS-18084-753714a3-81a7-cc37-5a95-de9e0459aa34 The influence of storage conditions and DNA extraction protocol on the results of molecular analysis of the European spruce bark beetle (Ips typographus L.) Vpliv postopkov shranjevanja in ekstrakcije celokupne DNA na rezultate molekularne analize osmerozobega smrekovega lubadarja (Ips typographus L.) 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. Za uspeh molekularne identifikacije podlubnikov je ključna zadostna količina kakovostnega ekstrakta DNA. Pri raziskavi vrste smo se osredotočili na vpliv različnih postopkov shranjevanja osebkov Ips typographus(L.) in različnih protokolov ekstrakcije DNA na količino in kakovost DNA, namenjene za uporabo v molekularni diagnostiki. Odrasle hrošče smo zamrznili pri -20 °C na dva načina, v etanolu in na suho. Ekstrakcije smo opravili po štirih različnih postopkih. Primerjali smo količino pridobljene DNA. Kakovost ekstrahirane DNA smo ocenili s polimerazno verižno reakcijo in sekvenciranjem po Sangerju. Način shranjevanja ni statistično značilno vplival na količino pridobljene DNA. DNA višje kakovosti smo pridobili iz hroščev, ki so bili zamrznjeni v etanolu. Le dva postopka ekstrakcije DNA sta dala DNA, uporabno za sekvenciranje. V količini pridobljene DNA se pri sekvenciranju uspešna postopka nista pomembno razlikovala. Določili smo optimalno kombinacijo postopkov shranjevanja v etanolu pri -20 °C in ekstrakcije celokupne DNA z ekstrakcijskim kitom Nucleospin Insect DNA proizvajalca Macherey Nagel, ki omogoča časovno učinkovito identifikacijo. early detection specimen storage total DNA extraction PCR polymerase chain reaction Sanger sequencing molecular diagnostics zgodnje zaznavanje shranjevanje osebkov ekstrakcijski postopek polimerazna verižna reakcija PCR sekvenciranje po Sangerju molekularna diagnostika true false false Angleški jezik Slovenski jezik Neznano 2024-02-02 10:59:02 2024-02-02 10:59:03 2024-02-07 13:41:03 0000-00-00 00:00:00 2023 0 0 str. 29-38 Vol. 132 2023 0000-00-00 Zaloznikova NiDoloceno NiDoloceno 0000-00-00 0000-00-00 2023-01-01 630*14:630*13(045)=111 2335-3112 10.20315/ASetL.132.3 180304131 https://doi.org/10.20315/ASetL.132.3 1 b392d059-c1b1-11ee-b0d4-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=24558 RAZ_Devetak_Zina_2023.pdf RAZ_Devetak_Zina_2023.pdf 1 4EBCAA87DBDD1FC548C5FED00E88F063 bb4ebfc66f1735e144c8daf9af2bcaff21b44486f67bd8afce49ebd915d08790 62ac5829-c1b2-11ee-b0d4-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=24560 Gozdarski inštitut Slovenije Acta Silvae et Ligni 0 0 0