1. Development of a multi-targeted real-time PCR assay for the detection of the grapevine pathogen Xylophilus ampelinusAleksander Benčič, Alexandra Bogožalec Košir, Janja Matičič, Manca Pirc, Neža Turnšek, Tanja Dreo, 2025, original scientific article Abstract: Background Xylophilus ampelinus is a plant pathogenic bacterium that causes bacterial blight in grapevines, which can lead to severe yield losses and economic damage. Owing to its fastidious growth on culture media, detection is primarily based on molecular methods. However, existing tests have produced inconsistent results, particularly when used to detect latent infections and non-validated matrices. There is a risk of false-positive results, with economic consequences such as restrictions on international trade. To enhance the diagnostics of X. ampelinus, a genome-informed approach was utilised to identify new potential targets for specific detection. On the basis of these sequences, multiple real-time PCR assays were designed, and their specificity and sensitivity were assessed, as well as their performance validated across three different grapevine tissues, including leaves, roots, and xylem. Results The newly designed real-time PCR assays were evaluated via high throughput testing for specificity and sensitivity and compared with a reference assay. The most promising assays were selected and validated in different grapevine tissues and included in a test performance study to validate their reproducibility and robustness. Three new assays (Xamp_BA_2, TXmp22.4, and Xamp_BA_7) demonstrated high specificity and sensitivity for X. ampelinus detection. The Xamp_BA_2 assay exhibited the best overall performance, offering high diagnostic sensitivity and robustness across diverse plant matrices. Importantly, the assays exhibited no cross-reactivity with non-target bacterial species and maintained high detection accuracy across diverse grapevine tissue types. Conclusions The newly developed real-time PCR assays provide an enhanced diagnostic framework for the detection of X. ampelinus in various plant matrices, significantly improving the applicability of molecular testing. The Xamp_BA_2 assay demonstrates superior performance and is recommended for routine diagnostics, with other validated assays being employed for confirmation of identification. The development of these new assays represents a significant expansion of our toolkit for the precise detection of X. ampelinus in grapevines, with the potential to contribute to the mitigation of grapevine bacterial blight, the prevention of yield losses, and the protection of international trade in grapevine material. Further implementation of these assays will support regulatory and phytosanitary efforts to mitigate the spread of X. ampelinus.
Keywords: Xylophilus ampelinus, grapevine bacterial blight, molecular diagnostics, Vitis vinifera, real-time PCR, genome-informed assay development
Published in DiRROS: 05.09.2025; Views: 373; Downloads: 162
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2. Test performance study on qPCR assays for detection of Phyllosticta citricarpaTjaša Jakomin, Janja Zajc Žunič, Polona Kogovšek, 2025, original scientific article Abstract: Citrus black spot (CBS), caused by the fungus Phyllosticta citricarpa, significantly affects citrus fruit marketability and can lead to premature fruit drop. Accurate and reliable detection of this quarantine pathogen is crucial, particularly for asymptomatic plant material. This study evaluated two qPCR assays, the EPPO recommended assay PC and assay Pc-TEF1, based on TEF region, for detecting P. citricarpa through a collaborative test performance study (TPS). DNA from the isolates of Phyllosticta spp. and other fungi was spiked into citrus fruit peel extracts (lemon, orange, and pomelo) and distributed among 13 laboratories. Sample and qPCR assay stability under typical transport conditions was confirmed, although prolonged storage affected Pc-TEF1 assay performance. The assays were assessed based on sensitivity, specificity, reproducibility, and repeatability. Both assays demonstrated high performance, with repeatability and reproducibility exceeding 95%. The PC assay, as expected, detected different related Phyllosticta species, while Pc-TEF1 showed higher specificity for P. citricarpa included in the TPS alone. Additionally, inhibitory effects were observed specifically in the pomelo peel samples, suggesting matrix-dependent variability. This TPS confirms that both PC and Pc-TEF1 qPCR assays are robust. Further evaluation of the qPCR assays would support the selection of the most reliable assays for the detection of P. citricarpa, contributing to the effective management of CBS disease in citrus production and trade.
Keywords: test performance study, Phyllosticta citricarpa, real time PCR, TEF1, biotechnology
Published in DiRROS: 07.05.2025; Views: 631; Downloads: 563
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3. One-step RT-droplet digital PCR : a breakthrough in the quantification of waterborne RNA virusesNejc Rački, Dany Morisset, Ion Gutiérrez-Aguirre, Maja Ravnikar, 2014, original scientific article Abstract: Water contamination by viruses has an increasing worldwide impact on human health, and has led to requirements for accurate and quantitative molecular tools. Here, we report the first one-step reverse-transcription droplet digital PCR-based absolute quantification of a RNA virus (rotavirus) in different types of surface water samples. This quantification method proved to be more precise and more tolerant to inhibitory substances than the benchmarking reverse-transcription real-time PCR (RT-qPCR), and needs no standard curve. This new tool is fully amenable for the quantification of viruses in the particularly low concentrations usually found in water samples.
Keywords: waterborne virus, quantification, droplet digital PCR, real-time PCR, digital PCR
Published in DiRROS: 04.03.2025; Views: 675; Downloads: 455
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4. Grapevine flavescence dorée (FD) follow up Vitisens, GRAFDEPI and Qdetect (GRAFDEPI2) : final reportMarina Dermastia, Helga Reisenzein, Luca Ferretti, 2015, final research report Abstract: 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.
Keywords: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Published in DiRROS: 16.09.2024; Views: 800; Downloads: 454
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8. Development of LAMP based protocol for accurate, reliable, fast and affordable diagnostics of Candidatus Phytoplasma solani : Euphresco success stroryMarina Dermastia, treatise, preliminary study, study Abstract: 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.
Keywords: phytoplasmas, grapevine yellows diseases, LAMP, real-time PCR
Published in DiRROS: 03.09.2024; Views: 896; Downloads: 579
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9. Retrospective survey of Dickeya fangzhongdai using a novel validated real-time PCR assayŠpela Alič, Katarina Bačnik, Tanja Dreo, 2024, original scientific article Abstract: 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.
Keywords: molecular testing, diagnostics, plant pathogen, real-time PCR, Dickeya, survey, water
Published in DiRROS: 07.08.2024; Views: 1008; Downloads: 656
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10. Assessment of the real-time PCR and different digital PCR platforms for DNA quantificationJernej Pavšič, Jana Žel, Mojca Milavec, 2016, original scientific article Abstract: 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.
Keywords: real-time PCR, molecular diagnostics, human cytomegalovirus, DNA quantification, digital PCR
Published in DiRROS: 25.07.2024; Views: 971; Downloads: 541
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