VITISENS IS A NEW DEVICE CREATED TO DETECT EPIDEMIC THREATS ON THE VINEYARDS CAUSED BY THE FLAVESCENCE DORÉE (FD) AND THE BOIS NOIR (BN) PHYTOPLASMAS.

1. Cost-Effective Hand-Held Device for Rapid In-Field Detection of
Flavescence Doree Phytoplasma in Grapevines

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 field
Contents Training Material
www.vitisens.eu

3. VITISENS project overview
http://www.isppweb.org/nlmar11.asp
www.flavescencedoree.org
Flavescence 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 moves
between vines to feed
The occurrence of FD disease
• Catastrophic in France and Italy.
• Incidence is rising rapidily in other EU countries in
recent years, showing a clear epidemic pattern of spread
• Straining grape and wine producers already
strugglying to maintain competitiveness with
regard to New Word and Asian producers.
www.vitisens.eu

4. VITISENS project overview
VITISENS proposes a portable handheld device answering to the need of wine producer
and wine plant nursery SMEs to perform rapid in-field tests to determine the presence of
the Flavescense Doree phyotplasma in grapevines by means of an innovative integration of
extraction, 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 diversity
shown in different grapevine varieties and due to the similarity with symptoms shown by other
physiological alterations or by diseases caused by other pathogens
VITISENS consortium believe that containment and even eradication of the disease is feasible but
only if timely and accurate detection of the FD is achieved and control measures are imediately
applied.
www.vitisens.eu

5. VITISENS project partners
SME´s Technical SME´s RTD performers
MERCIER
SAPRUEM
DALFONS
FORSITE
SEDIAG
FERA
CRIC
NIB
Click on the flags for more information
www.vitisens.eu

6. Scientific and Technological objectives
Scientific objectives: To fill the gaps in the current state-of-the-art in FD detection methods
while improving their specificity and sensitivity through the combination of molecular
methods and fluorescence detection technologies, as well as to innovate far beyond the
current laboratory detection methods.
Technological objectives: to develop a homogeneous, simple, rapid, accurate, hand held
and cost-effective diagnostic tool for detecting the Flavescence doreé phytoplasma with
cost below €1,000 per unit and €10 per test.
VITISENS device must integrate the three required steps to perform sensitive and accurate
detection of nucleic acids: extraction, amplification of specific sequences and detection.
www.vitisens.eu

7. Main Outcomes of the project. Market Survey
During the first 6 months of the project, a questionnaire was developed by the partners to
gather information on the market and end-users needs to define the final VITISENS
specifications.
In order to better understand and interpret the end-users answers, it was first necessary to
know if they were located in a region affected by Flavescence dorée or not. Among the
collected responses, it appears that 56% of the end-users questioned are based in regions
where FD is present.
Percentage of FD impacted or not end-users country by country
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8. Main Outcomes of the project. Market Survey
Money spent on fighting Flavescence Doreé
Flavescence dorée is a sensitive topic due to its quarantine status. Therefore end-users are
sometimes afraid either to communicate about FD in their vineyards or of the dissemination of
the 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 a
national survey of FD impact has been carried out in the past years and is updated every year. It
appears that 30 million € are spent in France to fight against FD (insecticide, material, labour
hours, etc…). Moreover, we also learnt that €150,000 were spent in 2010, only for Flavescence
dorée analysis.
Current detection method used, FD
testing?
56% of them do not perform tests
Notice: FD analysis are not
mandatory in regions not affected by
FD.
www.vitisens.eu

9. Main Outcomes of the project. Market Survey
Current detection method used, which FD detection/analysis?
Visual inspection of symptoms is the most common method used. Among
the end-users, more than 70% observe the symptoms and 33% send their
samples to laboratories.
When we focus on the end-users who declared doing FD tests, among the
71% 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-users
seem 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 soon
as possible, and over 90% of the end-users have to wait between 2
and 3 weeks for the results. That’s why 63 % of end-users think
that the current used method is not quick enough
www.vitisens.eu

10. Main Outcomes of the project. Market Survey
Price sensitivity, maximum price per test?
As the fight against FD is managed by governmental authorities, this is the
best source for global information regarding price of FD analyses.
60 % of the end-users doing FD testing pay between 50 and 100 Euros for a
test. 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 for
Flavescence 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 similar
facilities. 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 that
even if we propose a result of the analysis in 30 minutes, the running time of the analysis will be still
too long to perform all the tests in the vineyard. However, wine growers or nurseries will only want to
confirm visual symptoms on few samples, therefore, they prefer the in-field solution.
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11. Main Outcomes of the project. Market Survey
VITISENS important features
Conclusions
End-users located in the most FD-impacted countries were the most sensitized to the disease and the
need 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, the
analyses had been performed by legal authorities. When asked directly we found that 65% of the
questioned end-users thought that VITISENS would be useful to them.
www.vitisens.eu

12. Main Outcomes of the project
Work carried out during the project by RTD partners to develop the VITISENS
device
 Sample treatment protocol has been developed by NIB adapted to the field
conditions
 DNA extraction method was developed by CRIC specific for FD amplification
and further detection using laboratory new methods (LAMP) adapted for in field
conditions performed by FERA.
 Special instrumentation for optical detection and temperature control system
was developed by CRIC to carry out the tests
 CRIC has also design the portable device as well as the disposable cartridge
where the sample must be introduced.
VITISENS
Diagnostic
kit
VITISENS
Reader kit
www.vitisens.eu

13. Main Outcomes of the project
Innovative amplification method – Loop-Mediated Isothermal Amplification (LAMP)
Japanese company Eiken Chemical Co. Developed the novel nucleid acid isothermal amplification
method. The method has already been used under laboratory conditions for the detection of several
pathogens with highly successful and satisfactory results.
By defining four to six primers specifically for six to eight distinct regions on the target
gene, extremely high specificity is provided. By incubating all these reagents in a single
tube, sequences from either RNA or DNA are selectively amplified.
The amplification takes placed at constant temperature of 65ºC, therefore a heating stage is required
in the VITISENS reader.
Watch video
Eiken Genome Co., Ltd.
www.vitisens.eu

14. Main Outcomes of the project
VITISENS detection method – Optical detection
Fluorescence 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 emit
fluorescence when binding double stranded DNA, what means when the sample is infected. Emission
levels are measured by the VITISENS optical detection system and results are immediately showed on
the LCD screen.
Reaction chamber –
detection point
www.vitisens.eu

15. Main Outcomes of the project
Main advantages over Current State-of-the-Art
Limitation of SOTA VITISENS System
Assay heterogenity: various steps, different
devices required, sample manipulation
increasing of contamination risk
Single closed device with easy 2-step sample
preparation avoiding contamination
Platform costs: integrated platforms have a
market price over €5,000 (€50 per test)
Target price: €1,000 per device (€10 per
test)
Device portability: usually not hand held,
and often required a lap-top and main
power supply.
Portable, battery-powered handheld device
with LCD interface
Time required to perform the test: up to 3
days
Test results approximately in 40 min
Laboratory technicians required Design to be used by non-technical staff
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16. VITISENS Diagnostic Kit
264 mM Tris
236 mM Tris-HCl
137 mM NaCl
2 % PVP K-25
2 mM PEG 6000
0.05% Tween 20
Manual homogenization
Automatic homogenization
Two options (extraction tubes)
LAMP Primers (FD DNA Amplification)
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17. VITISENS Reader Kit
www.vitisens.eu

18. VITISENS Kit
Heating stage
Optical measurement stage
LEDs
On/Off Switch
Select button
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19. VITISENS Kit
Battery connection
Connection to
computer
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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 infected.
www.vitisens.eu

21. Sample collection
Time tissue amount
Spring flower 5 flower clusters
Spring / Summer Leaf vein 2 veins from 5 leaves
Summer / autumn berry 3-5 berries
 Sample on different parts of the plant
 If possible, take symptomatic tissue
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22. Sample preparation
1. Take 5 berries/flower clusters/leaves from different parts of the plant
• Tear/cut the leaf veins into small pieces
2. Add material to prepared tubes with buffer and beads/sand
• Berries to big tubes, leaf veins to smaller tubes
3. Mix for 2 minutes
4. Transfer 5 µl of extract to small tubes (dilution) and mix
5. Transfer 5 µl of the diluted extract to reaction
6 ml of
buffer
2 ml of
buffer
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23. Sample preparation
264 mM Tris
236 mM Tris-HCl
137 mM NaCl
2 % PVP K-25
2 mM PEG 6000
0.05% Tween 20
Manual homogenization
Automatic homogenization
www.vitisens.eu

24. Sample preparation
Transfer 5 µl of extract to small
tubes (dilution) and mix
Transfer 30uL of mixture
to the cartridge
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25. Validation data
Vitisens reader was tested on DNA, extracted from healthy, FD infected and BN
infected grapevine plants, and on homogenates prepared from healthy and FD
infected 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 homogenate
www.vitisens.eu

26. DISSEMINATION of VITISENS PROJECT
MATERIALS AND TOOLS ACTION / PRODUCT
VITISENS 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 as
vinegrowers.
Demonstration activities
(conferences, workshops,
courses)
Incorporate VITISENS results in activities already planned.
Other publications - Manuals
- Guidelines
- Good practice codes
- Protocols of use
VITISENS Mailing lists - Targeted mail shots to existing and new client networks /
Targeted audience database
www.vitisens.eu

27. Thank you for your attention!
For further information please visit: www.vitisens.eu
www.vitisens.eu

28. MERCIER FRÈRES S.A.R.L (MERCIER)
Mercier is a grapevine nursery that has been accumulating
for more than 100 years, a unique body of experience by
providing winegrowers with vines of the highest quality.
Presently, Mercier is the French leader in grafted vine
production with 20 million plants produced per year, half of
which being exported.
The nursery is part of the Mercier Group, which constitutes
a network of complementary businesses controlling the
entire production cycle and thus being very responsive to
customer demands.
Its current objective is not only to keep its leadership, but
also to increase its production by breaking into new
markets.
Contact person:
Mr. Olivier Zekri. R&D Manager
Website:
www.mercier-groupe.com
www.vitisens.eu

29. WEINGUT S.A.PRÜM (SAPRUEM)
S.A. Prüm Winery dates back almost 850 years. Today, it is
one of the most successful and famous wineries in Germany
(wines commercially produced for 200 years and distributed
worldwide to more than 50 countries), well known for
spectacular quality of Riesling grape white wines.
S.A. Prüm belongs to the VDP association (The Association
of German Premium Wineries), a group of privately-owned
wineries in possession of the most prestigious vineyards and
wine lands in Germany, with the goal of encouraging the
production of outstanding wines and maintaining the
identity and characteristics of a viticultural landscape and
environment, formed by unique vineyard parcels.
S.A. Prüm’s goal is to guarantee the integrity of the “Grands
Vins” and produce the highest available quality from top
vineyards with enormous ageing potential.
Contact person:
Mr. Raimund Prüm. General Manager
Website:
www.sapruem.com
www.vitisens.eu

30. CANTINE D’ALFONSO DEL SORDO S.R.L (DALFONS)
The D’Alfonso del Sordo family has been in the wine business since
1800, when they started winegrowing in northern Apulia (Italy).
Nowadays they are one of most dynamic and visionary Apulian
producers of fine wines, with estates concentrated in a suitable
viticultural area, and have played an important role in preserving
traditional oenological know-how.
DALFONS farm extends over 120 hectares in San Severo of which
90 hectares are vineyards. The company produces and bottles
wines only from its own vineyards. While a 30% of their annual
production remains in Italy, 70% is exported to European countries
as well as to other non-EU countries including the USA and Japan.
The company devotes a tremendous effort to improve the quality
of its wines, particularly focusing on safety aspects. Their company
strategy is strongly client-oriented, enabling the company to be
versatile in coping with market competition and consumer
demands.
Contact person:
Mr. Gianfelice d’Alfonso del Sordo.
General Manager
Website:
www.dalfonsodelsordo.it
www.vitisens.eu

31. FORSITE DIAGNOSTICS Ltd. (FORSITE)
Forsite Diagnostics specialises in the contract
manufacture of rapid immunoassays with a focus on
the human healthcare, veterinary and agri-food
sectors.
Forsite was established in 2007 as a spin-out from a
major UK government laboratory, and now has 14 staff.
The company operates from state-of the art facilities in
which are housed automated manufacturing equipment
for lateral flow assays.
Forsite has wide experience of working in national and
international projects, and is currently active in four EU
projects. Alongside expertise in assay development and
manufacture, Forsite has developed and market
launched the Pocket Diagnostic range of in-field tests
for plant pathogens.
Contact person:
Mr. Chris Danks. Chief Executive
Officer.
Website:
www.fositediagnostics.com
www.vitisens.eu

32. SEDIAG ANTICORPS ET DIAGNOSTIC SAS (SEDIAG)
Contact person:
Dr. Sam Seddas. President
Website:
www.sediag.com
The main activity of SEDIAG is the production, development
and marketing of reagents and diagnostic kits for various
agricultural and biotechnological applications.
Their list of customers includes public and private research
laboratories, plant diagnostic analysis labs, plant quarantine
stations, breeders, nurseries and food industries, in France
(50%) and internationally. Their products are present
worldwide thanks to their distributor network in more than
30 countries.
SEDIAG offers laboratory testing services for rapid and
reliable diagnostic assays in phytopathology and a large
range of ELISA reagents and kits to detect the presence of
major plant pathogens, especially in grapevines.
Recently, SEDIAG proposed a new service of custom
monoclonal antibody production.

33. The FOOD and ENVIRONMENT RESEARCH AGENCY (FERA)
Contact person:
Dr. Rick Mumford. Team leader.
Website:
www.fera.defra.gov.uk
FERA was established in 1992 as an executive agency of the
UK Department for Environment, Food and Rural Affairs
(DEFRA). It has a turnover of circa 60M € per annum and
employs about 700 staff in a new 200 million € state of the
art 1,000,000 m2 laboratory complex located 6 kilometres
outside the city of York.
It is a public sector science organisation providing research
and information services to governments and industry
around the world.
The Plant Health Group of FERA has pioneered the use of
molecular methods for rutine testing of quarantine plant
pathogens in support of statutory policy aims, including the
use of real-time PCR in the field situation and the use of real-
time PCR and microarray methods in the lab.
www.vitisens.eu

34. Centre de la Recerca i Innovació de Catalunya (CRIC)
Contact person:
Mr. Albert Nieto. Managing Director
Website:
www.cric.cat
CRIC is a Spanish multidisciplinary research centre which
assists small and medium sized enterprises to become
more competitive through technological
innovation, respecting the environment and improving
products, processes or services towards a better quality of
life.
Research activity is targeted toward electronic
sensors, mechanical engineering, process control and
instrumentation design, software and artificial
intelligence, telecommunications, chemical engineering
and biochemistry.
CRIC has participated in 35 EU funded projects in FP4, FP5
and FP6 and has acted as coordinator in many of them. It
has also participated in a number of national and regional
projects.
www.vitisens.eu

35. NATIONAL INSTITUTE of BIOLOGY (NIB)
Contact person:
Dr. Maja Ravnikar.
Head of the Biotechnology and System
Biology department.
Website:
www.nib.si
NIB is a public non-profit organization, with 130
employees, which performs basic and applied research in the
fields of biology, biotechnology, ecology and medicine. It is
certified as an ISO 9001 and ISO 17025 company.
The Department of Biotechnology and Systems Biology
participates in a large number of different research projects
dealing with plant pathogens, development of diagnostic
methods and biotechnology. Several projects include the
approaches of systems biology. The department researchers
are involved in EU FP7 funded projects
QBOL, Qdetect, VITISENS, Cytothret and GMO_SEEK; in many
COST actions and bilateral collaborations.
They closely collaborate with public and private companies, as
well as with governmental ministries. Lately a spin-out
company Biosistemika was established for offering complete
solutions in the field of molecular techniques.
www.vitisens.eu