Team:GreatBay SCIE/Human Practices
Integrated Human Practice Overview
Integrated Human Practice should always involve two-way dialogue - introducing a project to the wider public and getting their response in return. Integrated Human Practice is especially important for our portable toxin detection strip this year - since our ultimate aim is to (someday) commercialize our product and making it handy for people that pick mushrooms, we would have to better our product through meticulous planning and adequate conversation with expert mycologists and potential beneficiaries (people that pick mushrooms for a living, etc.). Thus, we devoted a lot of time and resources to contact all sorts of people, asking about their opinion of our project and receiving suggestions and ideas for improvements. Here is a detailed record of all our Integrated HP activities - who we talked with, what they got from us, what we got back from them.
Integrated Human Practice Overview Map.
Stakeholders
Talk with potential users -- Mushroomers
Yunnan Province, a fairytale place in China, is abundant in wild mushrooms. The local people there also have the habit of picking up wild mushrooms in the mountains and eating them. Also, some people will sell wild mushrooms in the market to make money. However, as some of the toxic mushrooms are almost indiscernible from non-toxic ones, some people would eat toxic mushrooms by mistake. According to Analysis on poisonous mushroom poisoning from 2010 to 2018 in Yunnan province, 9686 people were poisoned, 225 people died after eating toxic mushrooms. The case fatality rate was 2.32%, accounting for 57.30% of the total number of food poisoning cases, 30.37% of the total number of poisoning cases, and 50.00% of the total number of deaths in Yunnan Province from 2010 to 2018. In Yunnan Province, there are even more people who died of mushroom poisoning than people who died of COVID-19 from January to June 2020. Thus, we assume that people who live in places like Yunnan Province need a portable mushroom toxin detector which enables them to check whether the mushrooms they collect are toxic or not, thus reducing the chance that they eat toxin mushrooms by mistake.
1. Interview with a mushroomer in Yunnan Province
To further investigate the needs and concerns of mushroomers in Yunnan Province and improve our product, we have two rounds of interviews with a mushroomer through Wechat (a social media app used in China).
The interviewee is an experienced mushroomer in Yunnan Province and he loves to make videos about collecting wild mushrooms and upload them to public platforms. We are attracted by his videos and had an interview with him.
First round
During the first interview, we asked general questions about wild mushrooms and the habit of the mushroom collection in Yunnan Province. The mushroomer told us that it had been a tradition for local people in Yunnan Province to collect wild mushrooms for a long time as they were easily accessible in the hilly areas in Yunnan and they made the wild mushrooms into delicious dishes. Also, some mushrooms like Tricholoma matsutake and Collybia albuminosa cannot be artificially cultivated, so the wild ones are sold at a really high price, it can even reach 2000 RMB per kilogram.
Our chat history with the mushroomer and some poisonous mushrooms he picks.
However, he confessed that the only way for the mushroomers to discern toxin mushrooms from non-toxic ones is to discern by experience. However, there were too many different mushrooms and it was impossible for them to know whether each type of mushroom was toxic or not. As they had no portable equipment to check whether the wild mushrooms were toxic or not, so there was a possibility that they may eat toxic mushrooms and even sell them to other customers in the market, leading to public intoxication. Therefore, he suggested that we could make a cheap and portable mushroom toxin detector that is affordable to local people. According to his suggestion, we designed our product into a small detector that could easily detect mushroom toxins.
Our general hardware design.
Second round
During the second interview, we asked more specific questions about their concerns and expectations about the mushroom detector. He told us that he was concerned about the accuracy of the results given by the mushroom detector: he was afraid that if the wild mushrooms were toxic, but the detector's result showed that they were non-toxic, people who ate them may be poisoned. He told us that if we could make a toxic mushroom that could give accurate results, it would absolutely be a big hit and they could accept the price of it to up to 500RMB. Also, he was concerned about the time needed to detect the toxins in mushrooms: detection time within half an hour is acceptable for them.
2. Questionnaires for more mushroomers
In order to investigate more mushroomers' expectations about our product, we made an online questionnaire and disseminated it through various public platforms. The questionnaire is anonymous as we pointed out at the beginning of the questionnaire.
60% of the respondents of our questionnaire are male and 40% of them are female. All of them came from Yunnan Province, Guangdong Province, Jilin Province, and Liaoning Province which are mountainous and abundant in wild mushrooms. 26.67% of them are aged under 18, 60% of them aged between 18-55 and 13.33% of the, aged above 55.
At first, we investigated why they collect wild mushrooms. 60% of them said that they loved to eat wild mushrooms, 53.33% of them said that they were interested in mushrooms and 20% of them said that they could sell mushrooms. These reasons explain why though the government suggests people not to collect or eat wild mushrooms, many people are still fond of collecting and eat wild mushrooms. It also implicates that our mushroom toxin detector may be useful to the people who are fond of collecting and eating wild mushrooms by reducing the chance that they eat toxic ones.
60% of them thought it was common to eat wild mushrooms in their habitats. It shows that people in provinces that are abundant in wild mushrooms may eat wild mushrooms, therefore, they may eat poisonous wild mushrooms by accident.
Also,67% of the respondents said that they collect wild mushrooms more than twice a week in certain seasons which are abundant in wild mushrooms. Such a high frequency of collecting wild mushrooms may also increase the probability of collecting toxic wild mushrooms by accident. A majority of respondents(87%) also confessed that they had met toxic mushrooms while collecting mushrooms.
However, when asked how did they know whether the mushrooms they collect were toxic or not, 2/3 of them said that they discerned the toxic ones by "experience imparted by others" and over 1/2 of them said that they discerned by "personal experience". As a person could hardly discern all the mushrooms just by experience, there might be a chance that they could not discern some toxic mushrooms. Thus, our mushroom toxin detector may help them discern some unknown toxic mushrooms.
In order to investigate the mushroomers' requirements about the mushroom toxin detector, we asked questions concerning the mushrooms' priority about the detector, the price, and detection time they could accept.
The rank of the respondents' priority (from high to low) is accuracy -- portability -- detection time -- price -- safety -- appearance. It indicates that the respondents attached much importance to the accuracy and portability of the detector. The product needs to identify the toxic mushrooms correctly, so the aptamer for the toxins should have a high specificity so it will neither show negative results for the toxins we want to detect nor show positive results for non-toxic mushrooms. Also, as the mushroomers need to carry the detector when they collect wild mushrooms in mountains, they expect the detector to be portable, more specifically, light and occupy a small space. Thus, when we are designing our product, we should think about how to minimize the weight and size of the detector.
Moreover, 2/3 of the participants said that they could only accept a detection time up to 30 minutes, so we need to figure out ways to shorter the detection time. At last, 2/3 of them show that they could accept the price up to 500 RMB, hundreds of times lower than the existing ways to detection, thus, thinking about how to lower the cost is also essential.
3. Feedback on our Product
We showed a mushroom collector how to use our product and he was satisfied with it as it was cheap and portable. However, he mentioned that we could improve our product by putting the devices into a box so he could put all the devices together and carry them easily. We incorporated his suggestions and made a toolbox to put all the devices needed together to enhance the portability of our product.
Identify a new potential user -- researchers who need to collect wild mushrooms
1. Phone interview and offline meeting with Dr. Deng of Guangdong Institute of Microbiology
Dr. Wanqiu Deng is a mycology expert affiliated with the Guangdong Institute of Microbiology, where fungi and microorganisms are analyzed and studied. In order to find out more about how mushroom poisoning affects our local region - Southern China, we made a phone call with Dr. Deng, who happily informed us of rich, professional opinions.
"The reason why the majority of people would pick up and consume wild mushrooms is because of local traditions, and some just want to risk a taste of new things." ----Dr. Deng.
A group photo with Dr. Deng and other professors.
We have also acknowledged that the current methods are not good enough even within professional laboratories. Nowadays, the researchers are using HPLC and ELISA for quantitative and qualitative analysis/detection, but both of them are time-consuming, require professional researchers to operate, and inaccurate due to the use of antibodies. There is not a single completed device in the academic field that can provide rapid, easy, portable detection for the mushroom toxins in the whole world. Professor Deng spoke highly of our project, as our idea might be an answer to the blank in the market. Her words gave us the confidence to keep exploring down this track.
Later in August, we visited Guangdong Institute of Microbiology in Guangzhou, for further and deeper exchange of ideas and proposals with Dr. Deng and many other professors there. We presented our project, and the professors offered us suggestions on improving our project and loads of encouragement.
The mushrooms we collected on-site in BaiYun Mountain.
2. Mushroom Collection in Baiyun Mountain, Guangzhou
After visiting the Institute, we decided to go up to Baiyun Mountain, to explore the wildest parts of it under the leadership of a researcher from the institute, with the purpose of on-spot investigation of mushroom distribution on a regular mountain. This experience let us realize how easily wild mushrooms can be collected by people, and mushrooms are indeed difficult to be distinguished without professional guidance, and many times the professionals make mistakes as well. This trip made the purpose of our project more solid, and it had confirmed our aim of building a portable mushroom poison detection device.
3. A questionnaire for researchers as a potential user for the detector
After our visit to the Guangdong Institute of Microbiology, we identify another stakeholder group -- the researchers who need to collect wild mushrooms. In order to investigate their expectation about the mushroom toxin detector, we designed a questionnaire for them.
In order to identify the problems of the existing mushroom toxin detector machines, we asked about the respondents' opinions about the disadvantages of the machines. 75% of them thought that it was too expensive and the detection time was too long. 100% of them thought that they were unportable. Therefore, we need to pay attention to the price, detection time, and portability of the toxin detector.
Also, it is noticeable that when asked about the rank of priority of the detector, all of the respondents ranked "accuracy" as the most important one. Therefore, we need to pay attention to the accuracy of the detector when it is designed for the researchers.
At last, when we briefly described our detector, all of the respondents thought it would be very helpful for their researchers.
4. Feedback on our Product
We showed Dr.Deng from Guangdong Institute of Microbiology how to use our product and she provided us with useful suggestions. On one hand, she spoke highly of our product and said that it was a "portable, cheap and user-friendly" product. On the other hand, she suggested that it would be better if we could facilitate the entire design on a single component, using the microfluidic methodology to avoid cross-contamination. We adopted her suggestions into our hardware design.
Interview with Emergency Department doctor Ying Yang
Zoom interview with Doctor Ying Yang
Apart from mushroom collectors and the researchers, we also have an interview with another stakeholder group --- doctors who treat the patients poisoned by the toxic mushrooms.
From the interview, we acknowledge that an accurate judgment for mushroom poisoning cannot be achieved in the hospital. The detection of samples taken from patients needs to be sent to special detention centers to be done in a few days, and even this is not accurate enough. The doctors can only judge according to the patients' symptoms. This current situation further and more firmly illustrates that our project is meaningful, and could contribute very much to the market.
The doctor also said that if there was a detector to detect whether the patients' urine contains certain types of toxins and it has a short detection time, it will be really helpful for the treatment of patients by providing different treatments according to different types of toxins.
This current situation provided by the doctor confirmed again that our project is useful and meaningful, and could contribute a lot to the market and field.
Here is the video of our interview with Doctor Yang.
Experts in different fields
1. Interview with Dr. Candace Bever
Zoom interview with Dr. Candace Bever
Dr. Candace Bever is a Research Microbiologist who works in the Foodborne Toxin Detection Department of US Department of Agriculture. We cited some of her recent publications on LFIA amatoxin detection, and decided to turn to her for technical advice through a Zoom meeting.
Dr. Bever offered us suggestions on project design and hardware mechanism. She also relieved some of our unnecessary worries on toxin-related experiments' safety issues, and guided us with professional protection and disposal methods.
After acknowledging our technical difficulties in creating amanitin-BSA conjugates, Dr. Bever connected us with Professor. Yanru Wang, of Northwest University of Agriculture and Forestry Sciences, who happily provided amanitin-BSA conjugates for our SELEX experiments.
2. Interview with Dr. Ciara O'Sullivan and Dr. Miriam Jauset-Rubio
Our chat history with Dr. Ciara O'Sullivan and Dr. Miriam Jauset-Rubio
Dr. MiriamJauset-Rubio and Dr. Ciara K. O’Sullivan are the authors of Aptamer Lateral Flow Assays for Ultrasensitive Detection of β‐Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers，which inspires us to do our project. We adopted the RPA technique and the Competitive β-conglutin assay on the strip in this paper into our project.
We had a zoom meeting with them and they gave us a lot of precious suggestions on experiments and hardware design like how to design the test line and control line for our hardware, how long the complementary strand for the aptamer could be and how to calculate the dissociation constants of aptamers and toxins by building a model.
3. Interview with EPFL iGEM 2019
Zoom meeting with EPFL iGEM 2019
After receiving responses from literature authors of our core technical challenges, we thought it would be as useful if we took advice from past iGEMers. Therefore, knowing that the 2019 overgraduate Grand Prize winner team, EPFL, shared similarities in constructing hardware as well as integrated Human Practice with our project, we got in touch with them through email and they kindly offered us suggestions in our Zoom meeting.
They offered us technical advice on RPA and how to quantify the result using it. As we are both iGEM teams with RPA in our projects, they could give us the primary, first-hand source of information based on their very own experience as iGEMers - not researchers. EPFL 2019 has won the Special Prize for Integrated Human Practice, and they have suggested we establish a two-way dialogue to gain advice on our hardware from the stakeholders - in our case, mushroom hunters, researchers, and doctors. The meeting with EPFL also made us realize the importance of "turning an iGEM project into a start-up". There are actually many things to consider, for example, what kind of people would be our target user and in what ways should our hardware device be introduced to them.
4. Interview with Professor Strzałka
Our chat history with Professor Strzałka
Dr. Wojciech Strzałka is one of the authors of the Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides and we use the primers and the aptamers he selected for alpha-amanitin in our experiments.
We had a Zoom meeting with him and he kindly gave us some insightful suggestions. He suggested that we could calculate the enrichment constant after each round of SELEX to determine which round of SELEX has a higher percentage of aptamers which could bind to amatoxins.
Also, he told us that most of the recent researchers were based on alpha-amanitin, few were based on beta-amanitin and gamma-amanitin, so if we could select proper aptamers for beta-amanitin and gamma-amanitin, we could fill the gap.
5.Interview with Dr.Qinqin Han
Our chat history with Dr.Han
Dr. Qinqin Han is from Kunming University of Science and Technology and she specializes molecular diagnosis and biological detection, including the application of fast detection method based on aptamer technology of Nucleic acid.
She selected an aptamer H06 for alpha amanitin and applied Enzyme-Linked OligoNucleotide Assay (ELONA) and Dot blot to confirm the binding specificity of the aptamer. We utilized her methods to test the specificity of aptamer Best 1 and Best 2 (anotheraptamers for alpha amanitin selected by Professor Strzałka), However, we faced several difficulties and never succeeded until, thankfully, we successfully contacted Professor Qinqin Han through phone. She provided us with detailed suggestions about how to improve our experimental methods. After adopting her advice, we finally successfully confirm the specificity of aptamer Best 2, as well as our own aptamers.
Conference
CCiC - Conference of China iGEMer Community
The Conference of China iGEMer Community, CCiC, is one of the biggest academic meetings for iGEMers all around China to exchange and give out ideas through presentations and poster sessions. This year, we attended the 7th CCiC conference online, presenting our project through video presentations and posters. The judges and other teams approved our project, and we offered advice for other iGEM teams within our knowledge.
We won both awards in CCiC, as the only high school team among all winner teams - Best Presentation and Best Poster.