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Keywords: varstvo gozdov, morfološke analize, program preiskav, Fusarium circinatum, Pinus, borov smolasti rak, PCR
Published in DiRROS: 19.04.2024; Views: 12; Downloads: 0
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Keywords: varstvo gozdov, morfološke analize, program preiskav, Fusarium circinatum, Pinus, borov smolasti rak, PCR
Published in DiRROS: 19.04.2024; Views: 12; Downloads: 0
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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: 413; Downloads: 123
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Keywords: Geosmithia morbida, bolezen tisočerih rakov, diagnostični protokol, molekularna analiza, ekstrakcija DNA, PCR v realnem času, verifikacija
Published in DiRROS: 07.06.2023; Views: 325; Downloads: 0

Abstract: Since late 20th century DNA sequencing became the method of choice method in precision species identification. The ITS region is one of the official fungal barcoding DNA markers, although in some cases sequencing of the ITS re-gion may, due to misidentification, mislabeling or nomen-clature errors in public databases, lead to incorrect or insuf-ficient identification, as is currently a case in the genus Tu b e r. The aim of this study was to test, which ITS primer pairs are most appropriate and optimal for Tu b e r species DNA region amplification. Thereby we (1) compared ampli-fication success for different Tu b e r species using fungal spe-cific primer pair ITS1f and ITS4 and (2) compared amplifi-cation success using different ITS primer pair combinations in amplifying DNA region an example species Tuber aesti-vum. Based on results, Tuber aestivum was one of the most reluctant Tu b e r species in this study and in most cases failed to amplify with the above primer pair. After comparing dif-ferent ITS primer pairs, we conclude that the primer pair ITS5 and ITS7 is the most appropriate primer pair for ampli-fication DNA region of T. ae stiv um as it resulted in high am-plification success from ectomycorrhizal root tips. Based on sequences, gained from public databases, we found that ITS1f and ITS6 primers have a mismatch in one base pair compared to the target sequence of Tuber aestivum, thus re-sulting in poor or no amplification success. Although prim-er pair ITS5 and ITS7 in our study was proven to be the most appropriate primer pair in amplifying DNA region Tu b e r aestivum species, further analysis about appropriateness of it for a general barcoding and identification of ectomycorrhiza in complex community samples is needed.
Keywords: Tuber spp., ITS region, PCR amplification, ITS primers
Published in DiRROS: 30.07.2020; Views: 1593; Downloads: 1176
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