20.500.12556/DiRROS-19712 Filling the gaps in diagnostics of Pepino mosaic virus and Potato spindle tuber viroid in water and tomato seeds and leaves Waterborne and seedborne Pepino mosaic virus (PepMV) and Potato spindle tuber viroid (PSTVd) pose serious threats to tomato production due to seed transmission and mechanical transmission, coupled with their long-term stability outside the host plant. Therefore, rapid and sensitive diagnostic procedures are needed to prevent the spread of these quarantine pathogens. In particular, water and seed contamination are difficult to detect and confirm without efficient concentration methods. This study presents procedures that improve detection of PSTVd from tomato seeds and leaf tissue, and PepMV from water and tomato leaf tissue. For efficient concentration of PepMV from water samples, a procedure was optimized using convective interaction media monolithic chromatography columns, which provides concentration by three orders of magnitude. For concentration of PSTVd from seed extracts, an easy-to-use and efficient method was developed based on RNA binding to positively charged anion-exchange resin beads that provides up to 100-fold more sensitive detection in comparison with procedures without a concentration step. This thus allows confirmation of RT-qPCR results with sequencing of RT-PCR products in samples with low viroid levels. In addition, reverse-transcription loop-mediated isothermal amplification assays for detection of PSTVd and PepMV were optimized and adapted to both laboratory and on-site testing requirements. This allows rapid detection of these pathogens in crude leaf homogenates, in under 30 min. These procedures of concentration and detection are shown to be efficient and to fill the gaps in diagnostics of PepMV and PSTVd, especially when these pathogens are present at low levels in difficult matrices such as water and seeds. PSTVd PepMV seeds water concentration loop-mediated isothermal amplification true false true Angleški jezik Ni določen Neznano 2024-07-24 03:46:47 2024-07-24 15:24:07 2024-07-25 04:14:57 0000-00-00 00:00:00 2017 0 0 str. 1191-1201 issue 7 Vol. 66 2017 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2017-09-01 577.2 0032-0862 10.1111/ppa.12710 4302159 RAZ_Mehle_Natasa_2017.pdf RAZ_Mehle_Natasa_2017.pdf 1 6730A5CAAC1EFBF75A9F9A918923FCB1 1e7ba8d041e6e44e04c5dee163bfebb7250cdfcb76a3894454ffb114a0a35ad0 c8aa6272-49af-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27386 https://doi.org/10.1111/ppa.12710 0 9ab288ac-49af-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27385 Nacionalni inštitut za biologijo 0 0 0