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Povzetek: Background Detection and quantification of plant pathogens in the presence of inhibitory substances can be a challenge especially with plant and environmental samples. Real-time quantitative PCR has enabled high-throughput detection and quantification of pathogens; however, its quantitative use is linked to standardized reference materials, and its sensitivity to inhibitors can lead to lower quantification accuracy. Droplet digital PCR has been proposed as a method to overcome these drawbacks. Its absolute quantification does not rely on standards and its tolerance to inhibitors has been demonstrated mostly in clinical samples. Such features would be of great use in agricultural and environmental fields, therefore our study compared the performance of droplet digital PCR method when challenged with inhibitors common to plant and environmental samples and compared it with quantitative PCR. Results Transfer of an existing Pepper mild mottle virus assay from reverse transcription real-time quantitative PCR to reverse transcription droplet digital PCR was straight forward. When challenged with complex matrices (seeds, plants, soil, wastewater) and selected purified inhibitors droplet digital PCR showed higher resilience to inhibition for the quantification of an RNA virus (Pepper mild mottle virus), compared to reverse transcription real-time quantitative PCR. Conclusions This study confirms the improved detection and quantification of the PMMoV RT-ddPCR in the presence of inhibitors that are commonly found in samples of seeds, plant material, soil, and wastewater. Together with absolute quantification, independent of standard reference materials, this makes droplet digital PCR a valuable tool for detection and quantification of pathogens in inhibition prone samples.
Ključne besede: PCR amplification, inhibition, qPCR, droplet digital PCR, environmental samples
Objavljeno v DiRROS: 02.08.2024; Ogledov: 90; Prenosov: 68
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Povzetek: Currently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water bodies. Since exposure to as few as 10–100 particles of RoV or NoV may induce gastrointestinal disease, there is a need to develop a rapid and sensitive diagnostic method for their detection in coastal water samples. In this study, we evaluate the application of methacrylate monolithic chromatographic columns, commercially available as convective interaction media (CIM®), to concentrate pathogenic enteric viruses from saline water samples prior to virus quantification by one-step reverse transcription quantitative PCR (RT-qPCR). Using RoV and NoV as model enteric viruses, we present our results on the most effective viral concentration conditions from saline water matrices using butyl (C4) hydrophobic interaction monolithic support (CIM® C4). C4 monolithic columns exhibit a good capacity to bind both RoV and NoV and both viruses can be eluted in a single step. Our protocol using a 1 ml C4 column enables processing of 400 ml saline water samples in less than 60 min and increases the sensitivity of RoV and NoV detection by approximately 50-fold and 10-fold respectively. The protocol was also scaled up using larger capacity 8 ml C4 columns to process 4000 ml of seawater samples with concentration factors of 300-fold for RoV and 40-fold for NoV, without any significant increase in processing time. Furthermore, C4 monolithic columns were adapted for field use in an on-site application of RoV concentration from seawater samples with performance equivalent to that of the reference laboratory setup. Overall, the results from successful deployment of CIM C4 columns for concentration of rotavirus and norovirus in seawater samples reiterate the utility of monolithic supports as efficient, scalable and modular preparative tools for processing environmental water samples to enhance viral detection using molecular methods.
Ključne besede: rotavirus, norovirus, seawater, fecal contamination, qPCR, sewage
Objavljeno v DiRROS: 25.07.2024; Ogledov: 131; Prenosov: 63
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Povzetek: Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).
Ključne besede: next generation sequencing, small RNA, ribosomal RNA depleted total RNA, detection, plant viruses, plant viroids
Objavljeno v DiRROS: 25.07.2024; Ogledov: 106; Prenosov: 70
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Povzetek: High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato, Henbane mosaic virus (Potyvirus, Potyviridae), which was first discovered at the beginning of 20th century in the United Kingdom in cultivated henbane. A field tomato plant with severe necrotic symptoms of unknown etiology was sampled in Slovenia and high-throughput sequencing analysis using small RNA and ribosomal RNA depleted total RNA approaches revealed a mixed infection with Potato virus M (Carlavirus, Betaflexiviridae), Southern tomato virus (Amalgavirus, Amalgamaviridae) and henbane mosaic virus in the sample. The complete genomic sequence of henbane mosaic virus was assembled from the sequencing reads. By re-inoculation of the infected material on selected test plants, henbane mosaic virus was isolated and a host range analysis was performed, demonstrating the virus was pathogenic on several plant species. Due to limited metadata in public repositories, the taxonomic identification of the virus isolate was initially putative. Thus, in the next step, we used small RNA sequencing to determine genomic sequences of four historic isolates of the virus, obtained from different virus collections. Phylogenetic analyses performed using this new sequence information enabled us to taxonomically position Henbane mosaic virus as a member of the Potyvirus genus within the chili veinal mottle virus phylogenetic cluster and define the relationship of the new tomato isolate with the historic ones, indicating the existence of at least four putative strains of the virus. The first detection of henbane mosaic virus in tomato and demonstration of its pathogenicity on this host is important for plant protection and commercial tomato production. Since the virus was initially present in a mixed infection, and its whole genome was not sequenced, it has probably been overlooked in routine diagnostics. This study confirms the applicability of a combination of high-throughput sequencing and classic plant virus characterization methods for identification and phylogenetic classification of obscure viruses and historical viral isolates, for which no or limited genome sequence data is available.
Ključne besede: henbane mosaic virus, Tomato, high-throughput sequencing, host range analysis, phylogeny, Potyvirus
Objavljeno v DiRROS: 24.07.2024; Ogledov: 110; Prenosov: 103
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Povzetek: Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY’s flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA–coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein’s amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.
Ključne besede: plant pathogens, potato virus Y, viral RNA
Objavljeno v DiRROS: 23.07.2024; Ogledov: 137; Prenosov: 124
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