1. Grapevine flavescence dorée (FD) follow up Vitisens, GRAFDEPI and Qdetect (GRAFDEPI2) : final reportMarina Dermastia, Helga Reisenzein, Luca Ferretti, 2015, končno poročilo o rezultatih raziskav Povzetek: Europe is the world’s main producer and exporter of grapevine planting material and wine. This important economic sector is facing epidemic threats of at least 10 grapevine yellows diseases (GY) caused by phytoplasmas. In Europe the main phytoplasmas associated with GY are ‘Candidatus’ phytoplasma solani’ (BNp), which is a causal agent of bois noir and FDp, which causes flavescence dorée. Phytoplasmas are notoriously difficult to detect and identify and their specific detection relies exclusively on the molecular methods. Recently new methods, which are reliable, sensitive, fast, less expensive and suitable for using onsites, have been introduced. Among them is a loop-mediated isothermal amplification (LAMP) method, with several advantages (e.g. low sensitivity to plant extracts inhibitors, speed, robustness, simplicity of use) over the other methods (e.g. the real-time PCR). In a recently finished FP7 project VITISENS, a new LAMP protocols have been developed for specific detection of FDp, however, they have not been tested in the interlaboratories trials. In addition, there is no validated LAMP protocol available for the specific detection of BNp at the moment.
The main objectives of this project were:
(1) Development of new loop-mediated isothermal amplification (LAMP) based protocols for accurate, reliable, fast and affordable diagnostics of ‘Candidatus Phytoplasma solani’ (BNp), which will be applicable in-field
(2) To study new possible hosts plants and insect vectors of phytoplasma FDp.
(3) To organize an interlaboratory test performance study (TPS) to obtain validation parameters for the selected LAMP protocols for BNp, as well as for the LAMP assay for FDp detection developed in the course of the FP7 project VITISENS.
Ključne besede: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Objavljeno v DiRROS: 16.09.2024; Ogledov: 51; Prenosov: 27
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5. Development of LAMP based protocol for accurate, reliable, fast and affordable diagnostics of Candidatus Phytoplasma solani : Euphresco success stroryMarina Dermastia, elaborat, predštudija, študija Povzetek: Phytoplasmas are cell-wall-free plant pathogenic bacteria; they have a broad range of plant hosts and diseases of many important crops are associated with these pathogens. At least ten phytoplasma ribosomal subgroups have been associated with grapevine yellows diseases, which have great economic impact on viticulture. In Europe, the main phytoplasmas associated with grapevine yellows are the causal agent of flavescence dorée and ‘Candidatus Phytoplasma solani’, which cause bois noir.
Ključne besede: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Objavljeno v DiRROS: 03.09.2024; Ogledov: 103; Prenosov: 72
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6. Retrospective survey of Dickeya fangzhongdai using a novel validated real-time PCR assayŠpela Alič, Katarina Bačnik, Tanja Dreo, 2024, izvirni znanstveni članek Povzetek: Dickeya fangzhongdai, an aggressive plant pathogen, causes symptoms on a variety of crops and ornamental plants including bleeding canker of Asian pear trees. Historical findings stress the need for a specific detection tool for D. fangzhongdai to prevent overlooking the pathogen or assigning it to general Dickeya spp. Therefore, a qualitative real-time PCR for specific detection of D. fangzhongdai has been developed and validated. The developed assay shows selectivity of 100%, diagnostic sensitivity of 76% and limit of detection with 95% confidence interval in plant matrices ranging from 311 to 2,275 cells/mL of plant extracts. The assay was successfully used in a retrospective survey of selected host plants of relevance to Europe and environmental niches relevant to D. fangzhongdai. Samples of potato tubers and plants, plants from the Malinae subtribe (apple, pear, quince, and Asian pear tree) and fresh surface water from Slovenia were analyzed. D. fangzhongdai was not detected in any plant samples, however, 12% of surface water samples were found to be positive.
Ključne besede: molecular testing, diagnostics, plant pathogen, real-time PCR, Dickeya, survey, water
Objavljeno v DiRROS: 07.08.2024; Ogledov: 209; Prenosov: 151
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7. Assessment of the real-time PCR and different digital PCR platforms for DNA quantificationJernej Pavšič, Jana Žel, Mojca Milavec, 2016, izvirni znanstveni članek Povzetek: Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.
Ključne besede: real-time PCR, molecular diagnostics, human cytomegalovirus, DNA quantification, digital PCR
Objavljeno v DiRROS: 25.07.2024; Ogledov: 229; Prenosov: 137
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8. Accurate quantification and characterization of adeno-associated viral vectorsDavid Dobnik, Polona Kogovšek, Tjaša Jakomin, Nejc Košir, Magda Tušek-Žnidarič, Maja Leskovec, Stephen M. Kaminsky, Janet Mostrom, Hyunmi Lee, Maja Ravnikar, 2019, izvirni znanstveni članek Povzetek: One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron microscopy (TEM) for viral vectors’ characterization and quantification. Results of the analyses showed that droplet digital PCR (ddPCR) performs better than quantitative real-time PCR (qPCR), in terms of robustness and assay variance, and this was especially relevant for partially purified (in-process) samples. Moreover, we demonstrate the importance of sample preparation prior to PCR analysis. We evaluated viral structure, presence of aggregates and impurities with TEM analysis and found that these impacted the differences in viral titers observed by qPCR and ddPCR and could be altered by sample preparation. These results serve as a guide for the establishment of the analytical methods required to provide measures of identity and purity for AAV viral vectors.
Ključne besede: absolute quantification, AAV, gene therapy, electron microscopy, digital PCR, real-time PCR
Objavljeno v DiRROS: 23.07.2024; Ogledov: 236; Prenosov: 125
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9. Development and validation of a new TaqMan real-time PCR for detection of 'Candidatus phytoplasma pruni'Zala Kogej Zwitter, Marina Dermastia, Nataša Mehle, 2020, izvirni znanstveni članek Povzetek: Phytoplasmas of the 16SrIII group are wide spread, and have a broad plant host range. Among these, ‘Candidatus phytoplasma pruni’ (‘Ca. P. pruni’; phytoplasmas of 16SrIII subgroup A) can cause serious diseases in Prunus species and ‘Ca. P. pruni’-related strains can infect other plant species, including grapevines. In this study, a new real-time PCR detection system was developed for ‘Ca. P. pruni’ using TaqMan chemistry. This test was designed to detect ‘Ca. P. pruni’, by amplifying the species-specific secY gene. In addition, a test to amplify the group-specific 16S rRNA gene region was also developed. The performances of both tests were evaluated. The test that amplifies the secY gene provided reliable and quick detection of ‘Ca. P. pruni’. Using the newly developed and validated test, ‘Ca. P. pruni’ was not found in any of the 434 field samples collected from different plants species grown in different regions of Slovenia.
Ključne besede: phytoplasma, X-disease, real-time PCR, Prunus
Objavljeno v DiRROS: 22.07.2024; Ogledov: 205; Prenosov: 187
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10. Development and validation of a one-step reverse transcription real-time PCR assay for simultaneous detection and identification of tomato mottle mosaic virus and tomato brown rugose fruit virusAntonio Tiberini, Ariana Manglli, Anna Taglienti, Ana Vučurović, Jakob Brodarič, Luca Ferretti, Marta Luigi, Andrea Gentili, Nataša Mehle, 2022, izvirni znanstveni članek Povzetek: Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3’NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10−5–10−6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.
Ključne besede: real-time PCR, tomato mottle mosaic virus, tomato brown rugose fruit virus, leaves detection, seeds detections, performance criteria
Objavljeno v DiRROS: 16.07.2024; Ogledov: 217; Prenosov: 185
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