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Vitisens EU FP7 Project


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Vitisens EU FP7 Project

  1. 1. Cost-Effective Hand-Held Device for Rapid In-Field Detection ofFlavescence Doree Phytoplasma in Grapevines
  2. 2.  Overview of the project and objectives Presentation of the consortium Main outcomes/results Presentation of VITISENS kit and running a test VITISENS tutorial (optional)Menu and functionsHow to run a testSoftware and Firmware – Installation and useSample preparation in the fieldContents Training
  3. 3. VITISENS project overview Dorée phytoplasma• A unicellular organism that lives and grows in the sap of grapevines• Responsible for the most destructive phytoplasma disease of grapevines• Severely damages plant productivity• Transmitetd by the leafhopper Scaphoideus titanius, as it movesbetween vines to feedThe occurrence of FD disease• Catastrophic in France and Italy.• Incidence is rising rapidily in other EU countries inrecent years, showing a clear epidemic pattern of spread• Straining grape and wine producers alreadystrugglying to maintain competitiveness withregard to New Word and Asian
  4. 4. VITISENS project overviewVITISENS proposes a portable handheld device answering to the need of wine producerand wine plant nursery SMEs to perform rapid in-field tests to determine the presence ofthe Flavescense Doree phyotplasma in grapevines by means of an innovative integration ofextraction, amplification and detection steps.Current detection procedure: visual inspection• This approach can not detect latent infections.• Leaf symptoms are mainly evident in the summer and are often misleading due to the diversityshown in different grapevine varieties and due to the similarity with symptoms shown by otherphysiological alterations or by diseases caused by other pathogensVITISENS consortium believe that containment and even eradication of the disease is feasible butonly if timely and accurate detection of the FD is achieved and control measures are
  5. 5. VITISENS project partnersSME´s Technical SME´s RTD performersMERCIERSAPRUEMDALFONSFORSITESEDIAGFERACRICNIBClick on the flags for more
  6. 6. Scientific and Technological objectivesScientific objectives: To fill the gaps in the current state-of-the-art in FD detection methodswhile improving their specificity and sensitivity through the combination of molecularmethods and fluorescence detection technologies, as well as to innovate far beyond thecurrent laboratory detection methods.Technological objectives: to develop a homogeneous, simple, rapid, accurate, hand heldand cost-effective diagnostic tool for detecting the Flavescence doreé phytoplasma withcost below €1,000 per unit and €10 per test.VITISENS device must integrate the three required steps to perform sensitive and accuratedetection of nucleic acids: extraction, amplification of specific sequences and
  7. 7. Main Outcomes of the project. Market SurveyDuring the first 6 months of the project, a questionnaire was developed by the partners togather information on the market and end-users needs to define the final VITISENSspecifications.In order to better understand and interpret the end-users answers, it was first necessary toknow if they were located in a region affected by Flavescence dorée or not. Among thecollected responses, it appears that 56% of the end-users questioned are based in regionswhere FD is present.Percentage of FD impacted or not end-users country by
  8. 8. Main Outcomes of the project. Market SurveyMoney spent on fighting Flavescence DoreéFlavescence dorée is a sensitive topic due to its quarantine status. Therefore end-users aresometimes afraid either to communicate about FD in their vineyards or of the dissemination ofthe information given by them. This part of the questionnaire was not answered by the end-users.Nevertheless, useful information was obtained from governmental organizations in France, since anational survey of FD impact has been carried out in the past years and is updated every year. Itappears that 30 million € are spent in France to fight against FD (insecticide, material, labourhours, etc…). Moreover, we also learnt that €150,000 were spent in 2010, only for Flavescencedorée analysis.Current detection method used, FDtesting?56% of them do not perform testsNotice: FD analysis are notmandatory in regions not affected
  9. 9. Main Outcomes of the project. Market SurveyCurrent detection method used, which FD detection/analysis?Visual inspection of symptoms is the most common method used. Amongthe end-users, more than 70% observe the symptoms and 33% send theirsamples to laboratories.When we focus on the end-users who declared doing FD tests, among the71% who observe symptoms, 67% also test FD in laboratory.Current detection method used, main end-users?The main end-users performing tests are the inspectors themselves. The other significant end-usersseem to be the nurseries even if the number of tests seems to be quite low (just for import control).Current detection method used, time to results?Since the disease spreads quite rapidly, results are needed as soonas possible, and over 90% of the end-users have to wait between 2and 3 weeks for the results. That’s why 63 % of end-users thinkthat the current used method is not quick
  10. 10. Main Outcomes of the project. Market SurveyPrice sensitivity, maximum price per test?As the fight against FD is managed by governmental authorities, this is thebest source for global information regarding price of FD analyses.60 % of the end-users doing FD testing pay between 50 and 100 Euros for atest. As a rule, every interviewed person found it too expensive.Interest in a new detection method?Among the end-users questioned, 65% of them (within the high and the quite high interest levels)stated that it would be necessary to have a quick, cost effective and reliable field diagnostic test forFlavescence dorée.Where would you like to perform the test/diagnosis?Half of the end users answered directly “in the field” and the other half “in the office” or similarfacilities. Inspectors, due to specific prevention campaign, will have to test several samples in a field.For them, it is more convenient to collect all the samples and analyse them later. They estimate thateven if we propose a result of the analysis in 30 minutes, the running time of the analysis will be stilltoo long to perform all the tests in the vineyard. However, wine growers or nurseries will only want toconfirm visual symptoms on few samples, therefore, they prefer the in-field
  11. 11. Main Outcomes of the project. Market SurveyVITISENS important featuresConclusionsEnd-users located in the most FD-impacted countries were the most sensitized to the disease and theneed to control it.63% of the end-users doing FD tests thought that their current detection method is not quick enough.60 % of them paid between 50 and 100 Euro and found it too expensive, and for the others, theanalyses had been performed by legal authorities. When asked directly we found that 65% of thequestioned end-users thought that VITISENS would be useful to
  12. 12. Main Outcomes of the projectWork carried out during the project by RTD partners to develop the VITISENSdevice Sample treatment protocol has been developed by NIB adapted to the fieldconditions DNA extraction method was developed by CRIC specific for FD amplificationand further detection using laboratory new methods (LAMP) adapted for in fieldconditions performed by FERA. Special instrumentation for optical detection and temperature control systemwas developed by CRIC to carry out the tests CRIC has also design the portable device as well as the disposable cartridgewhere the sample must be introduced.VITISENSDiagnostickitVITISENSReader
  13. 13. Main Outcomes of the projectInnovative amplification method – Loop-Mediated Isothermal Amplification (LAMP)Japanese company Eiken Chemical Co. Developed the novel nucleid acid isothermal amplificationmethod. The method has already been used under laboratory conditions for the detection of severalpathogens with highly successful and satisfactory results.By defining four to six primers specifically for six to eight distinct regions on the targetgene, extremely high specificity is provided. By incubating all these reagents in a singletube, sequences from either RNA or DNA are selectively amplified.The amplification takes placed at constant temperature of 65ºC, therefore a heating stage is requiredin the VITISENS reader.Watch videoEiken Genome Co.,
  14. 14. Main Outcomes of the projectVITISENS detection method – Optical detectionFluorescence technique is used to detect the resulting product obtained from the LAMP reaction.Specific fluorescence dyes are used together with the LAMP reagents. These dyes will emitfluorescence when binding double stranded DNA, what means when the sample is infected. Emissionlevels are measured by the VITISENS optical detection system and results are immediately showed onthe LCD screen.Reaction chamber –detection
  15. 15. Main Outcomes of the projectMain advantages over Current State-of-the-ArtLimitation of SOTA VITISENS SystemAssay heterogenity: various steps, differentdevices required, sample manipulationincreasing of contamination riskSingle closed device with easy 2-step samplepreparation avoiding contaminationPlatform costs: integrated platforms have amarket price over €5,000 (€50 per test)Target price: €1,000 per device (€10 pertest)Device portability: usually not hand held,and often required a lap-top and mainpower supply.Portable, battery-powered handheld devicewith LCD interfaceTime required to perform the test: up to 3daysTest results approximately in 40 minLaboratory technicians required Design to be used by non-technical
  16. 16. VITISENS Diagnostic Kit264 mM Tris236 mM Tris-HCl137 mM NaCl2 % PVP K-252 mM PEG 60000.05% Tween 20Manual homogenizationAutomatic homogenizationTwo options (extraction tubes)LAMP Primers (FD DNA Amplification)
  17. 17. VITISENS Reader
  18. 18. VITISENS KitHeating stageOptical measurement stageLEDsOn/Off SwitchSelect
  19. 19. VITISENS KitBattery connectionConnection
  20. 20. Running a test using VITISENS device Sample collection Sample preparation Sample introduction in the cartridge In the VITISENS device: Enter the sample name Enter temperature and time settings Select “Perform test” Insert the cartridge in the Optical measurement stage → Base line will be defined Insert the cartridge in the “Heating stage” → Wait until the LED turns green Insert the cartridge in the Optical measurement stage → The amplification will be measured. The result will be stored and displayed on the device screen: Sample clean or Sample
  21. 21. Sample collectionTime tissue amountSpring flower 5 flower clustersSpring / Summer Leaf vein 2 veins from 5 leavesSummer / autumn berry 3-5 berries Sample on different parts of the plant If possible, take symptomatic
  22. 22. Sample preparation1. Take 5 berries/flower clusters/leaves from different parts of the plant• Tear/cut the leaf veins into small pieces2. Add material to prepared tubes with buffer and beads/sand• Berries to big tubes, leaf veins to smaller tubes3. Mix for 2 minutes4. Transfer 5 µl of extract to small tubes (dilution) and mix5. Transfer 5 µl of the diluted extract to reaction6 ml ofbuffer2 ml
  23. 23. Sample preparation264 mM Tris236 mM Tris-HCl137 mM NaCl2 % PVP K-252 mM PEG 60000.05% Tween 20Manual homogenizationAutomatic
  24. 24. Sample preparationTransfer 5 µl of extract to smalltubes (dilution) and mixTransfer 30uL of mixtureto the
  25. 25. Validation dataVitisens reader was tested on DNA, extracted from healthy, FD infected and BNinfected grapevine plants, and on homogenates prepared from healthy and FDinfected plants.Validation data:• Operation with the machine is user friendly• Time to reach the T 62 °C is 5 – 10min• The temperature is constant• Single use pipettes OK• Cartridges need to be improved (leakage of the mixture).• Problems with autofluorescence of plant
  26. 26. DISSEMINATION of VITISENS PROJECTMATERIALS AND TOOLS ACTION / PRODUCTVITISENS website Publication and downloading of documents like:- Brochures;- Newsletters;- Press releases;- Scientific publications.- Project sheets;- Final reports;- Other deliverables coming from the project;All other information.VITISENS partners websites Publication and downloading of documents like:- Brochures;- Newsletters;- Press releases;- Other deliverables coming from the project;- All other information.Article publication Publication in journals for specific target group, such asvinegrowers.Demonstration activities(conferences, workshops,courses)Incorporate VITISENS results in activities already planned.Other publications - Manuals- Guidelines- Good practice codes- Protocols of useVITISENS Mailing lists - Targeted mail shots to existing and new client networks /Targeted audience
  27. 27. Thank you for your attention!For further information please visit:
  28. 28. MERCIER FRÈRES S.A.R.L (MERCIER)Mercier is a grapevine nursery that has been accumulatingfor more than 100 years, a unique body of experience byproviding winegrowers with vines of the highest quality.Presently, Mercier is the French leader in grafted vineproduction with 20 million plants produced per year, half ofwhich being exported.The nursery is part of the Mercier Group, which constitutesa network of complementary businesses controlling theentire production cycle and thus being very responsive tocustomer demands.Its current objective is not only to keep its leadership, butalso to increase its production by breaking into newmarkets.Contact person:Mr. Olivier Zekri. R&D
  29. 29. WEINGUT S.A.PRÜM (SAPRUEM)S.A. Prüm Winery dates back almost 850 years. Today, it isone of the most successful and famous wineries in Germany(wines commercially produced for 200 years and distributedworldwide to more than 50 countries), well known forspectacular quality of Riesling grape white wines.S.A. Prüm belongs to the VDP association (The Associationof German Premium Wineries), a group of privately-ownedwineries in possession of the most prestigious vineyards andwine lands in Germany, with the goal of encouraging theproduction of outstanding wines and maintaining theidentity and characteristics of a viticultural landscape andenvironment, formed by unique vineyard parcels.S.A. Prüm’s goal is to guarantee the integrity of the “GrandsVins” and produce the highest available quality from topvineyards with enormous ageing potential.Contact person:Mr. Raimund Prüm. General
  30. 30. CANTINE D’ALFONSO DEL SORDO S.R.L (DALFONS)The D’Alfonso del Sordo family has been in the wine business since1800, when they started winegrowing in northern Apulia (Italy).Nowadays they are one of most dynamic and visionary Apulianproducers of fine wines, with estates concentrated in a suitableviticultural area, and have played an important role in preservingtraditional oenological know-how.DALFONS farm extends over 120 hectares in San Severo of which90 hectares are vineyards. The company produces and bottleswines only from its own vineyards. While a 30% of their annualproduction remains in Italy, 70% is exported to European countriesas well as to other non-EU countries including the USA and Japan.The company devotes a tremendous effort to improve the qualityof its wines, particularly focusing on safety aspects. Their companystrategy is strongly client-oriented, enabling the company to beversatile in coping with market competition and consumerdemands.Contact person:Mr. Gianfelice d’Alfonso del Sordo.General
  31. 31. FORSITE DIAGNOSTICS Ltd. (FORSITE)Forsite Diagnostics specialises in the contractmanufacture of rapid immunoassays with a focus onthe human healthcare, veterinary and agri-foodsectors.Forsite was established in 2007 as a spin-out from amajor UK government laboratory, and now has 14 staff.The company operates from state-of the art facilities inwhich are housed automated manufacturing equipmentfor lateral flow assays.Forsite has wide experience of working in national andinternational projects, and is currently active in four EUprojects. Alongside expertise in assay development andmanufacture, Forsite has developed and marketlaunched the Pocket Diagnostic range of in-field testsfor plant pathogens.Contact person:Mr. Chris Danks. Chief
  32. 32. SEDIAG ANTICORPS ET DIAGNOSTIC SAS (SEDIAG)Contact person:Dr. Sam Seddas. PresidentWebsite:www.sediag.comThe main activity of SEDIAG is the production, developmentand marketing of reagents and diagnostic kits for variousagricultural and biotechnological applications.Their list of customers includes public and private researchlaboratories, plant diagnostic analysis labs, plant quarantinestations, breeders, nurseries and food industries, in France(50%) and internationally. Their products are presentworldwide thanks to their distributor network in more than30 countries.SEDIAG offers laboratory testing services for rapid andreliable diagnostic assays in phytopathology and a largerange of ELISA reagents and kits to detect the presence ofmajor plant pathogens, especially in grapevines.Recently, SEDIAG proposed a new service of custommonoclonal antibody production.
  33. 33. The FOOD and ENVIRONMENT RESEARCH AGENCY (FERA)Contact person:Dr. Rick Mumford. Team was established in 1992 as an executive agency of theUK Department for Environment, Food and Rural Affairs(DEFRA). It has a turnover of circa 60M € per annum andemploys about 700 staff in a new 200 million € state of theart 1,000,000 m2 laboratory complex located 6 kilometresoutside the city of York.It is a public sector science organisation providing researchand information services to governments and industryaround the world.The Plant Health Group of FERA has pioneered the use ofmolecular methods for rutine testing of quarantine plantpathogens in support of statutory policy aims, including theuse of real-time PCR in the field situation and the use of real-time PCR and microarray methods in the
  34. 34. Centre de la Recerca i Innovació de Catalunya (CRIC)Contact person:Mr. Albert Nieto. Managing DirectorWebsite:www.cric.catCRIC is a Spanish multidisciplinary research centre whichassists small and medium sized enterprises to becomemore competitive through technologicalinnovation, respecting the environment and improvingproducts, processes or services towards a better quality oflife.Research activity is targeted toward electronicsensors, mechanical engineering, process control andinstrumentation design, software and artificialintelligence, telecommunications, chemical engineeringand biochemistry.CRIC has participated in 35 EU funded projects in FP4, FP5and FP6 and has acted as coordinator in many of them. Ithas also participated in a number of national and
  35. 35. NATIONAL INSTITUTE of BIOLOGY (NIB)Contact person:Dr. Maja Ravnikar.Head of the Biotechnology and SystemBiology department.Website:www.nib.siNIB is a public non-profit organization, with 130employees, which performs basic and applied research in thefields of biology, biotechnology, ecology and medicine. It iscertified as an ISO 9001 and ISO 17025 company.The Department of Biotechnology and Systems Biologyparticipates in a large number of different research projectsdealing with plant pathogens, development of diagnosticmethods and biotechnology. Several projects include theapproaches of systems biology. The department researchersare involved in EU FP7 funded projectsQBOL, Qdetect, VITISENS, Cytothret and GMO_SEEK; in manyCOST actions and bilateral collaborations.They closely collaborate with public and private companies, aswell as with governmental ministries. Lately a spin-outcompany Biosistemika was established for offering completesolutions in the field of molecular