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Revision as of 14:33, 20 October 2019
Integrated Human Practices
Integrated human practices were really important for us as they shaped our project. We wanted to be certain that our product would be useful and fully adapted to the end user.
To do so we contacted all the organizations impacted by the problem:
- Who they are
- What they do in regards with FD
- Their relation with our project
Agroscope is the Swiss centre of excellence for agricultural research, and is affiliated with the Federal Office for Agriculture.
They are responsible for testing all the infected leaves.
They gave us the technical requirements for the test and helped us classify the pathogens.
- Who they are
- What they do in regards with FD
- Their relation with our project
Swiss organisation that implements the measures for plant pest and diseases control at the regional level.
They recognise infected vine stock, send samples to Agroscope and follow the spread of the disease.
They taught us how to recognize symptomatic plants and supervised our sampling of infected leaves.
- Who they are
- What they do in regards with FD
- Their relation with our project
Cultivate grapevine to make wine.
Their plants are directly affected by the diseases, they recognize symptomatic vine stock and report it to the phytosanitary police.
They told us about the problems caused by these diseases and the slow-going diagnostic method.
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March: learning about the diseases from a winegrower 
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March: learning about disease management in Switzerland
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May: getting technical specifications for our test
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July: classifying the organisms
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July: getting insight on Microneedle patches 
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August: learning to recognise the symptoms and trying our extraction method in the field 
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August: discussing our project with a local winegrower
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October: Testing our prototype in a local winegrower
March 17-27th: Phone calls with winegrowers
Learning about the diseases from a winegrower
During the brainstorming stage of the project we talked with people outside of the science world to see how we could use synthetic biology to solve some of their everyday problems. One of them was Maxime Guérinot, a winegrower from Champagne, France. After explaining to him what iGEM was, we asked him if he had any problems he thought we could help with. He told us about 2 deadly grapevine diseases, Flavescence Dorée and Bois Noir, which need lab analysis to be diagnosed. He also explained that the current method to test for these diseases is fastidious: it takes weeks to get the results back because testing can only be performed in three labs in all of France. We now had a goal: to produce a rapid, decentralized method to test for Flavescence Dorée and Bois Noir.
March 25th: Phone call with Phytosanitary police
Learning about disease management in Switzerland
By then we learned about the existence of Flavescence Dorée and Bois Noir and had done some preliminary research on the two diseases. We wanted to know more about how they were dealt with in Switzerland, so we contacted the Swiss federal phytosanitary police. They talked us through the current diagnostic system and the actors involved in each step. They also gave us valuable information on the way the diseases spread in our region, the Canton of Vaud, as well as the number of tests they performed each year. Finally, they gave us the contact information of Santiago Schaerer from Agroscope, the local expert on phytoplasma-related diseases.
May 13th: Phone call with Agroscope
Getting the technical specifications for our test
Our call had 3 goals: to get some specific information on the diseases, learn more about the current molecular diagnostic methods and ask for the requirements for a new test. First, Santiago Schaerer gave us some general information on phytoplasma, the type of bacteria responsible for both diseases. He told us that the highest concentration of phytoplasma could be found in leaf stems or primary veins, so we had to adapt our extraction procedure to specifically target those regions. Next, we talked about the current methods for detecting Flavescence Dorée and Bois Noir. We learned that these methods were regulated at the European level by the European and mediterranean Plant Protection Organization (EPPO). For winegrowers to be allowed to use our test, it would need to accredited by the EPPO. This means we had to adapt our test to follow their guidelines, for instance by adding a positive control, among other things. Finally, we asked Santiago Schaerer about the technical specifications they would need us to follow for them to consider using our test. Up to that point we simply wanted to be able to detect Flavescence Dorée, but he told us that it was absolutely essential for them to be able to detect both Flavescence Dorée and Bois Noir. Instead of testing for only one disease, we now had to be able to do three tests at once: a control, a test for Flavescence Dorée and a test for Bois Noir. This meant that the amplification method we had chosen, Loop-mediated isothermal AMPlification (LAMP), could not be used as it is not multiplexable. We changed to Recombinase Polymerase Amplification (RPA), a method better suited to multiplexing.
July 12th: Phone call with Agroscope
Classifying the organisms
We decided early on that we wanted to test our detection method on actual infected leaves to have a proof-of-concept. That implied working directly with pathogens, so we contacted EPFL’s biosafety office to try to classify them. We found out that Candidatus Phytoplasma Vitis, the pathogen responsible for Flavescence Dorée, is a Risk Group 3 organism. Under normal circumstances that would indicate we would be unable to manipulate it in the laboratory. We talked about our problem with Santiago Schaerer, who is the biosafety officer at Agroscope. He told us that since phytoplasma cannot be cultured, and that we simply wanted to amplify short DNA sequences, the risk classification could be downgraded to Risk Group 2 activity. He then gave us the contact information for Michel Jeanrenaud, a colleague of his working at the phytosanitary police that could help us take some infected samples from the field.
July 19th: Conference on Microneedles by Prof. Zhen Gu
Getting insight on Microneedle patches
We had the chance the attend a conference at EPFL held by Prof. Zhen Gu, a professor at the department of Bioengineering at the University of California, Los Angeles. He invented and characterized the microneedle patch we are using for our DNA extraction step. During his talk, Prof. Zhen Gu went over the different applications of the microneedle patch, which taught us much more about the technology we are using. At the end of the conference, we talked with him about our project and how we were using the microneedle patches. We wanted to increase the odds of extracting phytoplasma DNA (which is unevenly distributed in the plant), and had thought of using the patch multiple times on the leaf, eluting the DNA between each extraction. He suggested applying the patch on different parts of the leaves in quick succession and only eluting the DNA at the end, since the patch loses its absorption properties once soaked in elution buffer.
August 21st: Visit of an infected vineyard with the phytosanitary police
Learning to recognise the symptoms and trying our extraction method in the field
We met with Michel Jeanrenaud, a phytosanitary police officer, in an infected field about 30 km from our campus. He started by showing us infected grapevines and teaching us how to recognize the symptoms. He went on to explain how fighting against the diseases is done on 2 fronts: • In containment areas: Flavescence Dorée is known to occur so the appropriate measures are already in place. • In non-infected areas: new outbreaks have to be detected quickly so as to quickly set up a containment area and take the appropriate measures. We then explained our project to him. During that discussion it became clear that our final product would be perfectly suited to the second case scenario, i.e. to quickly detect new outbreaks. That led us to thinking about who the final user of our product would be. The phytosanitary police was the logical choice, but a single phytosanitary police officer is responsible for hundreds of hectares. This meant there would be a delay between the recognition of the symptoms by the winegrower and the diagnostic. To reduce that delay, we chose to make our kit useable by winegrowers. To make that possible, we started designing a prototype that would eliminate all pipetting steps in our test. At the end of the visit, Michel Jeanrenaud offered to supervise our sampling of infected leaves. We took some leaves to bring back to the lab, but most importantly we were able to test our extraction method directly on infected leaves in the field.
August 29th: Conversation with the owner of an infected vineyard
Discussing our project with a local winegrower
We visited Basile Neyroud, a winegrower in Chardonne, Switzerland, and asked him a few questions about the impact Flavescence Dorée and Bois Noir had on his vineyard. We were particularly interested in his opinion because his vineyard is located at the epicenter of a Flavescence Dorée outbreak. He told us he had to destroy a whole parcel of his vines because the disease had already spread too far. He seemed interested in trying out our test: he stated that if he had a quick diagnostic tool he could use on his own, he would be able to quickly remove the infected plant, eliminating the source of a possible new outbreak before it could take place.