Team:SJTU-BioX-Shanghai/Human Practices
“The power of a team can hardly cope with so many setbacks in a project. Therefore, we need to help each other and learn from each other.”
From the establishment of the project, the practice of ideas, the social needs to the final formation, our products are inseparable from a lot of help. With the help of government agencies and teachers, we finally determined “Detection Blitz” after a series of ups and downs. After the company and the market research, we firmed up our ideas. In the specific experiment process, we also got many useful suggestions and new tips from the laboratory. In order to achieve the maximum convenience and precision, we gathered different feedback and improvement opinions on the products through the investigation and the company inquiries. Finally, in the discussion and communication between different teams, we chose the most optimized pipeline and completed the construction of our project.
Thank the people and groups who have helped us, and thank the organizing committee, and hope we could use our achievements to give back to the society, and use the concept of “local teams to solve local problems” to do everything that we can to promote the progress of the world.
After consulting the teachers, we found that either the detection of a single target have already had a simple and rapid detection program, or it may be difficult to carry out with greater toxicity, or there is no need for detection; although this disillusioned many of our ideas, it also inspired us to conduct non-singularity testing for a wider range of substances.
1. Agricultural Product Safety Center
There have also been many food and drug safety accidents in the world, affecting the hearts of the public. In order to understand the hidden dangers of major safety accidents, we visited the Agricultural Products Safety Center.
Gain
We first learned about a series of policies and measures to ensure the safety of agricultural products. For example, control at the source, and only allow relevant agricultural personnel to purchase permitted low-toxic pesticide products; secondly, daily random inspections of fruit and vegetable products, aquatic products, meat and dairy products are performed, and HPLC-MS and other instruments are used for Non-targeted detection. However, these operations can only ensure food safety every week, and cannot prevent harmful products from flowing into the market in time before a hazardous accident occurs.
Therefore, it is necessary for the grass-roots departments to conduct rapid testing before the vegetable market starts to sell. Therefore, the rapid testing of pesticide residues is very important. We have learned that there is not only this demand in the market, but the government will also conduct a special bidding process to fund the company's continuous technological innovation. For example, we found that the functional paper-based microfluidic technology developed by Shanghai Ocean University can display the results within 4 minutes during the interview; Nanjing XiangZhong Technology Co., Ltd. uses rapid biochip inspection technology to automatically analyze the results by software. The detection process is completed in 3 minutes; Yirui uses a colloidal gold high-throughput detector to make a portable small handheld device to complete a 10-minute rapid detection; RuiXin has greatly reduced the cost of detection through thin-layer chromatography.
In summary, we have learned the importance of quick inspection in ensuring food safety, and learned that the repeated inspections of various pesticide residues can ensure the peace of mind of local people at the dinner table.
2. Wang Guoquan
3. Zhao Yanbin and Zhang Kun
The three interviews on disease, environment, and agriculture gave us a deeper understanding of the needs and existing problems of the local area, inspired our thinking, and provided social significance for the realization of the system.
1. Luo Hong
2. Shanghai Ruixin Technology Co., Ltd.
3. Yan Yaxian
Through the above-mentioned exchanges, we finally determined the significance and feasibility of the project, and strengthened our ideas, and detection Blitz was born. A series of professors we interviewed later provided instructive help to our specific experiment process.
Target screening steps:
1. Zhao Yanbin and Zhang Kun
2. Shen Jiefeng
3. Qian Xiuping
Experimental guidance:
1. Tang Zhuo
2. Cui Daxiang
3. Bai Linquan
4. The author of DRIVER
6.27 Email the author
We introduced our project design to the author——adding two extra sequences on the 5' and 3' end of the aptamer-coupled ribozyme, but we were not sure whether the two added sequences would influence the structure and function of the aptamer-coupled ribozyme, so we wanted to get some useful suggestions. The author recommended not adding the 5' and 3' sequences into the selection because it might make the loop back oligo too long. The loop back oligos for reverse transcription and ligation should be synthesized with PAGE purification to only get molecules that are exactly full length, and this is critical to the ligation efficiency and success of selection.
Our methods：not add the 5' and 3' sequences into the selection, and model to validate two added sequences would not influence the structure and function of the aptamer-coupled ribozyme.
7.16 video conference
Since our detection system works outside the cell, we are worried that aptamer-coupled ribozyme will self-cleave before it works. The author said that it is feasible to test while transcribing, as long as we add the sample to be tested when transcribing.
For the preparation of the ligand group, the author recommended adding a few more small molecules. Pay attention to the solubility of the small molecules and avoid chemical reactions with each other. The dissolution can be promoted by adding DMSO and adjusting the pH.
In addition, the author also mentioned that the cleaving time can be adjusted to reduce false positives caused by some aptazymes not finishing cutting. We also designed related experiments to find the optimal cutting time of aptamer-coupled ribozyme.
7.29 Email contact
We read the literature again and confirmed the operation of several detailed steps to the author, which is very conducive to our subsequent experiments.
8.10 video conference
We asked the author if there is a better quality control method. The author recommended using qPCR to measure the ratio of cleaved to uncleaved, but qPCR is easy to go wrong, so we should be careful. It is recommended to do a PAGE every few rounds to observe the strip length to ensure that the system works normally.
8.17 video conference
We used qPCR for quantification and found that the results were contrary to what we expected——the cleavage with target was higher than without target. We were puzzled about this and wanted to get some help from the author. The author stated that T4 connection is the most critical and the least efficient, and suggested that we increase the amount of T4 enzyme.
In the next few days, we try to increase the efficiency of T4 ligation, eventually figured out that our T4 ligase activity was inhibited by dATP, and the selection process worked as expected when we reduced the amount of dNTP to a final concentration of 0.25mM(0.0625mM each) in the reverse transcription system.
In addition, we sent the small molecules that we plan to screen to the author, hoping to get the author's suggestions. We got a very important message: aptazymes for posotively charged molecules containing N ,whose ring is relatively flat, are easier to screen successfully. Based on this, we updated our screening targets to select small molecules that are flat, contain N, and positively charged.
8.21 Email contact
Talking our new idea with the author——We hope to start with 10 to 20 rounds of -ligand selection at first in order to remove those sequences with no cleaving activity first. In the remaining library, we then repeat +ligand selection rounds to pick out those sequences that have no cleaving activity when binding with our ligand. We hope the interested sequences will come out more quickly in this way and it might be a better choice since our time is quitely limited. The author recommends not doing more than 3-5 rounds of -ligand selections because it might bring loss of library diversity. So we have to abandon this approach and proceed in accordance with the protocol.
8.27 Email contact
In the context of the Covid-19 epidemic, it is crucial to find new rapid virus detection methods, especially for responding to future public health events. Based on this, we asked the author whether we can screen for proteins, and we wanted to use the S protein of the new coronavirus as one of our screening targets. Author thought it's possible to try protein molecules, and it should work. She suggested not mixing the protein and the small molecules together though. It's generally easier to get protein aptamers than small molecules, so if the tube contains a mixture of proteins and small molecules, we'll probably only end up with the protein sensors. After getting the author's reply, we quickly put the S protein into our system for screening.
9.16 Email contact
To improve the effiency of cleavage and reduce false positive results, we tried a strategy of stopping the transcription (by heating at 95℃ for 2min and then cooling to 25℃ at 0.5℃/s) and then giving the ribozyme about 10 minutes to cleave at room temperature. However, this disposal didn't bring about much difference. The author told us at higher temperatures the ribozymes structure might also be disrupted so that wouldn’t necessarily help cleavage. So we had to find new method to improve the effiency of cleavage and reduce false positive results.
5. TU Delft
6. SJTU Software
7. Song Jie
In order to comprehensively improve our experimental results and produce practical products, we conducted on-site interviews and surveys and obtained a series of recommended measures.
In order to fully understand the field work experience of the quick inspection station and the application scenarios of our products, we conducted on-site visits and investigations to seven agricultural product markets in Shanghai, and obtained a lot of useful information.
Survey design:
Something about Quick-checking
At a time when food was short, environmental pollution and other issues are frequently occurring, rapid detection provides a convenient, rapid, and popular detection method for preliminary screening and detection of major pollution accidents. As the front-line staff, they are the most powerful Speakers.
Our gains:
During the inquiry and investigation, we learned that the rapid inspection station has strict requirements for the detection of pesticide residues and clenbuterol. They can detect pesticide residues as frequently as once a day, while meat and aquatic products are randomly inspected every two to three days. This means that there is a greater demand for rapid detection products .
Secondly, their products are updated more frequently, and there is competition in this market. Therefore, our rapid development of methods will help products continue to optimize and improve, and seize market opportunities.
Thirdly, most of the training personnel who carry out the experiment operation are not well-educated, and even if they are trained, they cannot guarantee the accuracy of the experiment operation 100%. Therefore, we have developed the idea of simplifying the operation of the experiment. And after trying, decided to use TMSD technology, by completing the transcription step at the central station, and then transported to the base station, which greatly reduced the difficulty of operation, also prevented irregular experimental operations, and successfully shortened the detection time to 10 minutes.
We believe that the joint guarantee of safety is not only in our technological innovation, but also in the support of policies and laws. Through the workshop of the New Pollutant Alliance, we put forward our own views on relevant policies, laws and regulations, and hope to put forward practical improvement plans through research and contribute to the progress of the local community.
Environmental Law:
With the rapid development of industry and the mass production and use of various chemicals, the harm of some new pollutants to public health and the ecological environment is gradually emerging. The Fifth Plenary Session of the Nineteenth Central Committee of the Communist Party of China clearly stated that "emphasis on the treatment of new pollutants." Taking environmental hormones, antibiotics, perfluorinated compounds, microplastics and other typical new pollutants as pioneers, we will promote the construction of China's environmental risk prevention system and the improvement of environmental quality management systems, and further promote environmental management to be scientific, precise, and systematic. It is an important strategic task during the "14th Five-Year Plan" period and a very important development direction in the future.
Due to the characteristics of new pollutants, compared with conventional pollutants, domestic and foreign countries face many common challenges in their treatment. New pollutants are not easy to degrade, easy to bioaccumulate and enrich, and it is difficult to study the mechanism of harm, transformation and migration. There are many types, large quantities, and wide distribution, involving a wide range of industries and a long industrial chain, but the unit product usage is small, the content is low in the environment, and the distribution is scattered, and its production, use and environmental pollution base are not easy to understand. It can be migrated over long distances, and its management requires coordinated prevention and control of large-scale areas. Some are newly synthesized by humans, with excellent product characteristics, and its substitutes and alternative technologies are not easy to develop.
In the long run, the treatment of new pollutants should be effectively incorporated into full-life-cycle environmental management with the goal of preventing ecological environmental risks and improving the quality of the ecological environment. During the "14th Five-Year Plan" period, focusing on domestic and foreign research hotspots and the focus of management and control of international conventions and frameworks, using typical EDCs, antibiotics, per fluorinated compounds, etc. as breakthroughs, focusing on regulations and systems, investigation and evaluation, standard setting, basic research, and restoration demonstrations , Capacity building and other links, consolidate the foundation of management and control, and strengthen the governance system and governance capacity building.
At the same time, improve laws and regulations. Promote legislation on environmental management of chemical substances. Promote the formulation of emission standards for key industries related to the production and use of typical new pollutants, environmental quality standards, and related product standards. Study and establish a chemical substance full life cycle environmental management system framework, environmental risk assessment and economic and social impact assessment system, damage compensation system, information disclosure and public participation system, information reporting, data collection and data quality supervision system, and further improve the existing advantages Controlled substances management system and environmental access system for new chemical substances.
Pesticide Regulatory Policy:
The existing supervision system is quite complete. The Food Safety Law and the Measures for the Supervision and Administration of the Quality and Safety of Food and Agricultural Products Market Sales clearly stipulate that the operators of edible agricultural products wholesale markets should be equipped with inspection equipment and inspectors, sampling inspection or rapid inspection frequency; Sellers purchasing edible agricultural products shall check relevant certification materials.
At the same time, retail market operators and sales companies are encouraged to equip with inspection equipment and inspectors, or to entrust a qualified food inspection agency to carry out sampling inspections or rapid inspections of edible agricultural products. It is understood that Zhejiang, Guangdong, Sichuan, Hunan, Yunnan and other provinces have carried out the "You Point and I Check" activities.
But there are still shortcomings. As shown in the figure below, with the rapid development of the logistics industry, e-commerce of agricultural products has gradually become an important part of the acquisition of agricultural products. We advocate to let agricultural products go out through the Internet, but the supervision of them has become a big problem.
Therefore, we propose to establish a special and rapid agricultural product transportation logistics line, and conduct random inspections and quick inspections of online products to ensure the safety of agricultural products during transportation.
At the same time, we are highly concerned about the products transported at home and abroad, and timely detection of possible new pollution and pesticides, and you can also choose to use our platform to develop new pesticide express kits!
