Overview
Our project focuses on the detection of antibiotic residues in the environment and antibiotic- resistance genes from the patients’ samples. In doing so, we seamless integrated human practices into our project advancement. We contacted 10 people from different areas, including researchers, doctors, farmers and government inspectors to learn about their individual requirements, ask for suggestions to improve our design. For instance, our product involves an integrated, automatic operation to save labor as the hospitals are always lack of hands. We also separated our detection process into two reaction steps, from pressing version to the final electromagnet version based on their suggestions, which helps make our module portable and prevent the contamination. We also added extra tubes to expand detection range and increase volume capacity of detection. After our initial design, we demonstrated it to potential users, the officials from CADCP and doctors we interacted before. We received valuable ideas to improve my design. Integrated Human practices played a significant role in our project, shaping our project from an idea into a product for the potential customers.
Intro
This is a story about pursuing a dream. Our dream is to minimize the antibiotic resistance situation worldwide. The starting point of this dream is very trivial: it may be that we need to take more medicine when we catch a cold or fever, taking longer to recover; it may be a higher cost for us to buy latest medicines since they are the only ones working. But the problem it might lead to can be life-threatening: the abuse of antibiotics will accelerate the formation of bacterial resistance. The existence and reproduction of bacteria with drug resistance in the environment will invalidate the treatment of countless diseases and make people vulnerable. In extreme cases, it can even force the society to return to the pre-antibiotic era. Since there’s no way we will tolerate to sit still, watching the crisis happening doing nothing, we must take actions and do our utmost to solve the problem of antibiotic resistance. This is our journey, are you ready to go on the road with us?
Preparation stage
First of all, before embarking on a project, we need to have a full and comprehensive understanding of it. For example, we need to investigate the causes and mechanisms of antibiotic resistance thoroughly, together with what harm it will cause to us humans. (More detailed information of our background research can be found here in our Description page: Description
Antibiotic resistance refers to the characteristics of some microorganisms that are highly resistant to antibiotics they are supposed to be sensitive to. The production of this tolerance is mostly due to the mutation of microorganisms, which can be regarded as a survival of the fittest in the microbial world. However, people’s abuse of antibiotics has accelerated the development and evolution of pathogenic microorganisms, forming more and more powerful ‘super bacteria’, making it more and more difficult for originally effective antibiotics to kill them. Many diseases have become incurable, casting vast shadows to the existing medical system
In order to further understand the problem, we need to know the frontline battlefield, the animal husbandry industry and our medical hospital. Therefore, we consulted professionals from Center for Animal Disease Control and Prevention and hospitals for a thorough analysis
1) Shandong Provincial Center for Animal Disease Control and Prevention
According to the data we gathered, one major source of antibiotic resistance in the environment is the animal husbandry industry. Hence, we contacted Mr. Yungang Li, Shandong Provincial Center for Animal Disease Control and Prevention, for their current methods in detecting antibiotics and what challenges they may face.
Mr. Li brought up a field problem. He mentioned that when their technicians collect the samples in the field, they will have to bring back all samples back and send them to specific laboratories for testing. They hope to have a quick test on site. The convenience and efficiency of current testing methods are not satisfactory at all.
Therefore, to save all the trouble, we shall focus on how to make a device cheap and convenient for outdoor use. After we told Mr. Li our idea, he spoke highly of our creativity and hoped that we could reduce the current testing cost through more advanced technology.
2) Tongji Hospital of Tongji University
The hospital is one of the major places for antibiotics usage, we then went to Tongji hospital to visit its laboratory and interview Dr. Zhiying Zhang.
According to Dr. Zhang, there are methods available to determine the specific resistant genes, such as mass spectrometer and real-time PCR. But the process is not fast and not very convenient in community hospitals. For example, in order to amplify the signals, sometime the bacteria-containing samples need to be cultured, which takes at least 6 hours. Furthermore, only larger hospitals can own this kind of expensive machines. Community hospitals and hospitals from less-developed regions have barely no access to such costly testing devices, thus resulting in the inequality problems between different areas.
Figure 1. Dr.Zhang being interviewed by us.
As a result, we decided to fix these two existing problems, to accelerate the speed of those examinations and to reduce the testing costs. These goals will help gain doctors’ time for treating precious life and may help gaining equality between people’s from different areas.
Brainstorming Stage
Which angle should we start from to solve this problem? After brainstorming, we believe that we should focus on monitoring the ‘cause’ and ’consequence‘ for antibiotic resistance. Only by grasping the head and the tail can the problem be completely solved.
We focused on the ‘cause’ of antibiotic resistance is antibiotic overuse/abuse. Therefore, the very first step toward solution is to efficiently detect the existence of antibiotic molecule. Efficient detection will guide the use for antibiotics in hospitals, help animal husbandry industry to limit the use, and help organizations/government to control or regulate the use. With convenient, cheap, and fast detection methods, it eventually will fundamentally improve the situation in antibiotic resistance.
The ’consequence‘ means the formation of bacteria drug resistance. We imagine that with robust detection methods for bacteria drug resistance genes, doctors will be able to choose proper antibiotics to save life timely. This will essentially reduce the total use antibiotics, especially the broad-spectrum antibiotics prescribed by doctor’s guessing. In order to carry the two concepts into our project, we need to find biological tools with the following properties:
In order to complete an integrated project that combines the two concepts we designed, we need to find some biological tools with unique characteristics to support it. We hope that after using these biological tools, our solution can have such properties:
1) It needs to be cheap and very cheap
As we want to solve the problem on a large scale, we must ensure that our device can be accepted and adopted by many people, and the first priority to that is a cheap price. Our product should not require high cost of purchase or storage and promote the equality of medication between imbalanced economic areas.
2) Its signal must be very readable
We hope that the results can be detected clearly, intuitively, and conveniently, such as using a fluorescent reporter to report the test results.
3) It needs to promise high biological safety
Out of full bio-safety considerations, we hope that our solution can avoid the use of in vivo gene editing and other methods, so as to avoid genetic contamination or other unexpected situations.
4) Its sensitivity must be very high
Our inspection results must be accurate, but in real life, we are likely to encounter insufficient samples. So there should be a stage of amplification before the start of the formal reaction to bring the sample concentration to an appropriate level to facilitate our detection.
5) It should be made into a portable mode
Because there are many cases we need to bring our designed devices to outdoor use to reduce intermediate transportation and storage management costs, the method we choose cannot be too sensitive to environmental temperature, light and other conditions, nor can it be affected by disturbances such as shaking and bumps.
6) It should own comprehensiveness
According to the plan we designed, we need to control the antibiotic molecules at the source and finally determine the existence of antibiotic resistance genes, so we need a ‘key’ that can open different locks to meet our vision and solve our problems. That is to say, our biological tool should be of high comprehensiveness, being able to complete various types of missions.
Ask for help
After determining the use of Cas12a as a major tool to complete our goal, in order to further understand its characteristics, and to better operate and use it in experiments, we consulted Dr. Quanjiang Ji, School of Physical Science and Technology, ShanghaiTech University. Dr. Ji is an expert in antibiotic-related research and Cas12a.
After discussion, Dr. Ji agreed with our initial concept:
1) Dr. Ji said that our intention coincidentally agreed with his long-existed concern. The problems we are considering are indeed significant, and the drug resistance caused by the abuse of antibiotics is poisoning our environment and medical system. Many hospitals have encountered the problem of using antibiotics without gaining the result they were expecting. Our products can help hospitals to resolve urgent needs.
2) Meanwhile, based on his relevant experience, Dr. Ji said that Cas12a is indeed a very good tool, which can identify DNA efficiently and accurately, meeting our needs.
Besides, Dr. Ji gave us very good advice on:
1) Adopt cell free reaction system to ensure efficiency and biological safety.
2) Some instructions on how to design cRNA.
Figure 2. Dr. Ji and our team members.
As for the reporter involved in the experiment operation, we chose to consult Dr. Xingxu Huang, School of Life Science and Technology, ShanghaiTech University. We pitched our idea to Dr. Huang, receiving positive feedback. However, there was still concerns.
Dr. Huang told us that the cutting efficiency of Cas12a for single strand sequence is highly sequence dependent. Therefore, it is critical to select the appropriate ssDNA and develop the relevant reporter.
1)Dr. Huang gave us some suggestions for choosing ssDNA.
We adopted Dr. Huang’s advice and tried out several sequences until choosing the TTATT.
Figure 3. Dr. Huang when being consulted.
Verification and improvement
Part One
Up till now, we have fully prepared for the experiments. After hard working together, we finally got the corresponding success and had feasible experimental results. We built aptamer-sensing module, Cas12a-reporting model, creating a system of our own.
With such results, we wanted to know whether it really solved the existing problems and whether there was room for improvement. For the sake of epidemic prevention, we contacted Dr. Wei Li, Qilu Hospital of Shandong University online and introduced our products to him, hoping to get some suggestions and feedback.
Dr.Li first expressed his appreciation and affirmation of our products, and believed that we had a good understanding of current problems and also actively assumed social responsibilities. However, he believes that if our products need to be put into actual production and use in hospitals, we were required to improve it. The suggestions for improvements he gave are as follows:
1) If possible, make our product semi-automatic and integrative. Only simple operation is required, and the sample can be automatically amplified after adding in, and then the reaction and detection can be performed by itself. This will reduce the shortage of manpower in the hospital and make our product more practical.
2) We need to use high-throughput methods to detect more accurately and cover a variety of existing examples more comprehensively to improve the feasibility of detection. The advice given by Dr.Li is as follows: More common antibiotic resistance genes:
i) NDM
ii) ESBC
iii) KPC
iv) OXA
The more important and dangerous ones are:
i) MCR
After collecting such feedback and precious information, we have made corresponding improvements to our products.
1) We have combined our products and designed a device that uses the 'press' method to combine the amplification reaction part and the detection part. By pressing, the two parts can be connected simply and conveniently, reducing the workload and operational complexity.
2) In order to solve the problem of throughput, we designed a set of tubes to detect different substances in different test tubes so as to cover the widest possible possibilities.
Part Two
After having the 1.0 version of the prototype implemented by the 'press' method, our team conducted self-analysis and discussion, and raised the problem of the existing prototype, that is, it is easy to cause pollution of parts, and many need to be replaced frequently, which rises the cost and wastes resources. So, we changed the prototype design in order to solve this problem. We modified it to include a centrifuge design, hoping to solve the problem of fusion of different parts through centrifugation.
With the latest centrifuge system, we went to Tongji Hospital of Tongji University and talked about our project with Dr. Zujun Sun of the Department of Laboratory Medicine and got improvement opinions and suggestions from him.
Figure 4. Dr. Sun bring to answer our questions.
1) The centrifuge is not good enough as a prototype mode. If the manual mode is used, the efficiency is not enough. If the electric mode is used, it needs to be connected to the power source because it needs to reach a certain power, leaving it to only be used as a fixed facility, and there is no way to meet mobile and portable requirements. Therefore, after careful consideration, we chose the electromagnet model machine. Once the sample is added into the tubes with RPA or aptamer, it will be amplified at a constant temperature. After a certain period of time, the electromagnet will cut off the power to let the metal piece holding Cas12a fall into the reaction system for related measurement.
In order to verify whether the metal piece enclosing the sample can well meet the needs of mixing our sample and the reacting system, we carried out modeling verification. After complex modeling proved, we found that this method can meet the demand well.
2) Dr. Sun believes that it is not accurate enough to pass the experimental results through our existing fluorescence characterization. It is better to have precise measurement of the reaction results, that is, the quantitative reality of the results of antibiotic resistance genes we measured. Correspondingly, we designed a fluorescence detection instrument to digitally express the reaction results.
Part Three
After that, we used the electromagnet type reaction model we designed to carry out repeated testing experiments and achieved very good results. So, we made videos of using this model to test samples and played them to doctors of Tongji Hospital of Tongji University, getting very positive feedback. At the same time, the doctor suggested that we contact with some lower-level hospitals to see whether the product fit them.
Therefore, we contacted Dr. Liping Wu, the 2nd Affiliated Hospital of Chengdu Medical College to inquire about the demands of some smaller hospitals.
Dr. Wu said that if their hospital has patients’ samples to be tested, they must be transported to a cooperating laboratory for further analysis operations, which is a very troublesome and time-consuming process. The emergence of our products can solve the problem of their incapability of detection and greatly help the clinics and patients. During the online communication, they watched us perform an operation and expressed their hope that our products can be put into production and used in actual clinical practice to help them with medical assistance.
Figure 5. The outlook of CESAR.
Part Four
In order to cater the need of detecting antibiotics, we did a whole lot of research to find an appropriate method and material. After this process, we finally aimed at aptamer, which was understood as a useful ‘hook’, turning molecule signal into DNA signal. We then used this aptamer-sensing module to replace the former RPA amplification part, constructing a new system with our existing Cas12a-reporting module and fluorescence-measuring module.
With a model that can detect antibiotic molecules, we re-contacted Mr. Yungang Li, Shandong Provincial Center for Animal Disease Control and Prevention to see if the products we designed could meet their needs.
After listening to our explanation, they said that the problems of portability and accuracy have totally been solved, but they often used a larger sample volume, and hope that we could adjust the volume of the model and reaction system.
So, we adjusted the size accordingly, and then in order to conduct a field inspection, we visited Liyuan Farm, a chicken farm, interviewing its owner Mr. Fei and conducting field trials.
According to Mr. Fei, in recent years, laws and regulations have strictly controlled the use of antibiotics, and almost no drugs containing antibiotics are allowed to be used. At the same time, the Center for Animal Disease Control and Prevention is also very sensitive to the use of antibiotics. It will conduct random checks each 1-2 weeks to see if there is any problem of antibiotic abuse. Therefore, they mainly focus on the automation and scientificization of breeding and reduce chicken’s diseases rate by choosing high-quality chicken species.
At Liyuan Farm, we gathered chicken feed on the spot to see if it contains antibiotics. The running of the test was very gratifying, and the chicken feed used by Liyuan met the standards. We were very pleased to have such a result.
Figure 6. The inside of the chicken farm.
Figure 7. Mr.Fei
So far, we have formed a very complete and systematic solution, we named it CESAR——Cas12a-based efficient solution of antibiotic resistance. We are using our own effort to accomplish CESAR, a new application that will fit in the blank of the examination of antibiotic resistance and quickly identify antibiotic molecules at scene. We have faith in CESAR, which is the way to stop the worse of antibiotic resistance, the key to a higher standard of medical operation, and a strong weapon to fight against powerful pathogen. With its obvious merits, like cheap cost, high efficiency, portability and convenient operation, CESAR will definitely change the world.
We hope that its birth will be a milestone on the arduous road to solve the problem of antibiotic resistance and there will finally be no more ill-treated diseases. Everything will be better, starting today.
