Browsing by Subject "RNA-hiljennys"

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  • Jukkala, Jaana (Helsingin yliopisto, 2019)
    Multiplex PCR uses several primer pairs, each specific for different DNA sequences in the same reaction, enabling viruses from different families and genera to be recognized by the same test. The new broad-based method of detecting plant viruses, siRNA analysis, is expected to improve the identification of plant viruses as it does not require a prediction of viruses that may be present in the sample. Due to the method, limiting the test to detecting only certain viruses is unnecessary. The aim of this study was to optimize two or more single-phase multiplex RT-PCR tests for the pest laboratory of the Finnish Food Safety Authority (now known as Finnish Food Authority). The tests were for nine published, degenerate primer pairs for the simultaneous identification of Potyvirus, Potexvirus, Tobravirus and Tospovirus genera, Tobamovirus subgroup 1, Nepovirus subgroups a and b, Bromoviridae family and Carmovirus, Dianthovirus, and Tombusvirus, which belong in the Tombusviridae family. Both multiplex RT-PCR tests and siRNA deep sequencing were used to detect plant viruses from infected samples or samples showing viral symptoms. By identifying the viruses in the plant samples, the goal was to estimate the suitability of multiplex RT-PCR tests for identifying multiple RNA viruses from different genera. siRNA analysis was used to ensure the correctness of the multiplex RT-PCR results. In this study, two multiplex RT-PCR tests were created to detect viruses belonging to eight different virus groups. The obtained results serve as a basis for the validation of the multiplex RT-PCR tests. The validation is required to verify the suitability of the method for its intended use and the reliability of the results of the method. The results showed that siRNA deep sequencing was able to detect almost the whole genome from some of the found viruses. Multiplex RT-PCR tests detected Lupine mosaic virus (LuMV) from lupine (Lupinus polyphyllus Lindl.), Arabic mosaic virus (ArMV) from Chinese astilbe (Astilbe chinensis (Maxim.) Franch.) and from quinoa (Chenopodium quinoa Willd.), Tobacco rattle virus (TRV) from moneywort (Lysimachia nummularia L.) and possibly a new Potyvirus species from honey clover (Melilotus albus Medik.). siRNA analysis detected the same viruses, which increases confidence in the optimized multiplex RT-PCR tests.
  • Streng, Janne (Helsingfors universitet, 2013)
    RNA silencing is a sequence specific RNA degradation mechanism which is used by plants to regulate gene expression and to combat virus infections. However, viruses have developed so called silencing suppressors, which can prevent and interfere silencing reaction by many ways. For example, virus proteins can bind to maintaining proteins of the silencing reaction or to molecules which are responsible for signaling of the silencing reaction. This thesis focused on the study of protein-protein-interactions between known silencing suppressors of crini- and potyviruses and four maintaining plant proteins of RNA silencing. Protein-protein-interactions were studied using the yeast two-hybrid system (YTHS) and the bimolecular fluorescence complementation assay (BiFC). The latter method enables visualization of the studied protein interactions in plant cells. Protein expression of the cloned genes in yeast vectors were studied by using western blot. BiFC analysis was focused on protein interactions which were found by YTHS. This study detected three previously unknown protein interactions. Two virus proteins were found for the first time to bind directly to silencing maintaining proteins that are known to be targets of other silencing suppressors. Because the functions of these silencing maintaining proteins are known, it is possible that the three interactions described in this study interfere RNA silencing by impeding the functions of the plant proteins.