Abstract
The number of drug-resistant bacteria in the ocean has increased at an alarming rate over the past decade due to environmental pollution, the misuse of antibiotics and other factors. If the development of drug-resistant bacteria is not curbed, these "superbugs", which are inherently difficult to eliminate, will become one of the biggest global health threats in the next 30 years. The impact of drug-resistant bacteria on humans has suddenly become real. This problem will now affect millions of people directly or indirectly. It is an urgent problem that we need to be aware of immediately.
Hainan University is located on the shore of the largest ocean in China. The utilization and sustainable development of Marine environmental resources have always been the focus of our research. The HainanU_China team 2022 is dedicated to solving the problem of detecting drug-resistant bacteria in the ocean.
Interview with Experts
(Click picture and you can learn details)
Figure 1a.Professor Liao
In order to understand the main methods of bacterial nucleic acid detection at present, we asked Professor Liao Chenghong, director of the Department of Biotechnology, Hainan University.
Q: What are the main methods of nucleic acid detection at present? What are the characteristics of each method?
A: There are four main methods: PCR, thermostatic amplification, sequencing and CRISPR.
PCR method means that fluorescent substances are added to the reaction system, real-time fluorescence detection is realized by special instruments, and quantitative calculation of the template is carried out. It is the most commonly used nucleic acid detection method at present, and generally only takes 2 to 3 hours to get the test results.
The constant temperature amplification method avoids the temperature adjustment of the sample used in fluorescent PCR. The thermostatic device can complete amplification and chromatography detection, which is suitable for lower-level medical institutions and various application scenarios to realize POCT detection of pathogen RNA.
Sequencing can directly read the sequence of the nucleic acid bases, which is necessary for a new virus that is completely unknown. However, only a few companies have developed and put into production nucleic acid sequencing kits for the novel coronavirus.
CRISPR's detection technology is a new type of RNA detection that takes only a purified sample of nucleic acid molecules and can be detected in just one hour in three simple steps. At the moment, it's not very common.
Based on the teacher's guidance, we believe that each of the four methods has advantages and disadvantages, and the CRISPR test is more attractive to us -- it is convenient, fast and currently uncommon.
Figure 1b. Professor Zhou
Later, we interviewed Professor Zhou Hailong, who was more familiar with CRISPR and viruses. Q: What are the methods of nucleic acid detection?
A: Nucleic acid detection mainly includes nucleic acid detection reagent and antibody detection. The first method is usually PCR detection through throat swab, gene amplification, and then nucleic acid detection. And the antibody test is the choice of blood drawing, blood drawing method to get the results, observe whether the antibody titer is increased, this situation often refers to the situation that can not produce antibodies before blood drawing.We still prefer noninvasive testing, so we ruled out antibody testing.
Q: What is the specific process of CRISPR assays currently available?
A: At present, Professor Zhang Feng's team has realized the application of CRISPR-CAS system to complete the detection of COVID-19. Using synthetic 2019-NCoV RNA fragments, his team designed and tested two guide Rnas (grnas), each of which specifically recognizes a characteristic nucleic acid fragment of 2019-NCoV. When combined with the Cas13 protein, they form a SHERLOCK system capable of detecting the presence or absence of 2019-NCoV viral RNA. You can check Professor Zhang Feng's papers and so on.
Q: Are there any drawbacks to CRISPR-based nucleic acid testing?
A: There are also drawbacks. Although the CRISPR-CAS system based pathogen nucleic acid detection has the advantages of fast speed, low cost and strong practicality, its cost is too high, and the accuracy and sensitivity need to be systematically verified in clinical practice.
Figure 1c. Dr. Wan Yi
Combined with the teachers' answers, we preliminarily believe that we can design a nucleic acid detection instrument with higher accuracy based on CRISPR-CAS system.
On this basis, we contacted Dr. Wan Yi from the State Key Laboratory of Marine Resources Utilization in the South China Sea, Hainan University.
Q: Do you have any suggestions for the direction we should take?"
A: In terms of the direction of the project, I think the location advantage of Hainan is A very important concept, and your direction is also in line with this concept. The south China sea as China's four largest and deepest sea field, the area with the most abundant natural resources, the resources for our country, on the economy, national defense has important strategic significance, and the drug resistance problem of the Marine microorganisms can seriously affect people's production and living, national, and even the whole world there is no doubt that this problem in the south China sea is a problem that cannot be ignored in this mysterious treasure.
Q: Do you have any comments on the microbial resistance test?
A: The increasing drug resistance of pathogenic microorganisms threatens the safety of human production and life. Rapid detection, quantitative analysis and combined with antimicrobial management are key interventions to control the emergence and spread of drug resistance. However, at present, phenotypic antimicrobial susceptibility testing (AST) is more commonly used in clinical microbial resistance detection, most of which are broth microdilution method, disk method and so on. These methods need longer incubation time, which cannot guide anti-infection treatment in time. The drug resistance gene detection method based on the nucleic acid detection technology of CRISPR⁃Cas system can greatly shorten the reporting time, which is conducive to more targeted follow-up work according to microbial resistance.
In terms of project experiments, CRISPR/Cas13a, as a ribonucnitase, can only recognize and cut RNA. The nucleic acid amplification product to be tested must be transcribed in vitro to generate RNA before it can be recognized and cut by Cas13a, and Cas14 belongs to type ⅴ CRISPR system. At present, Cas14 family members including Cas14a to Cas14c have RuvC nuclease domain, which is responsible for binding to crRNA and tracrRNA and cutting target DNA, and can recognize and cut ssDNA. So I suggest that you use these two effector proteins for the purpose of detecting RNA and DNA resistant microorganisms
Study and summary of previous teams
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Figure 2a. CU-Boulder
CU-Boulder
（https://2014.igem.org/Team:CU-Boulder）
Introduction: Brittle cell 9 consists of an endonuclease (Cas9) that is directed by the CRISPR RNA component to a specific sequence in the genome. Once targeted to the genome, Cas9 causes double-strand breaks that kill host bacterial cells. Because killing depends on the sequence of the CRISPR-guide RNA, the sequence can be engineered to contain short specific nucleotide sequences and therefore target unique genes in any disease-causing bacterium.
Using non-replicating phages as vectors, we can efficiently deliver the CRISPR-Cas9 machinery to cells and kill bacteria by targeting the genome with CRISPR-Cas9.
Learning: They used CRISPR-Cas9 phages to enter cells, and the bacteria expressed Cas9 endonuclide and guide RNA (gRNA). The guide RNA consists of a spacer sequence that binds DNA and a handle bound by Cas9 endonuclease. Cas9 protein and gRNA are clustered together. Cas9 binds to the PAM site, allowing gRNA to anneal to the target sequence. Upon successful binding, Cas9 endonuclease cleaves DNA, causing double-strand breaks. If the cell is unable to repair the damage on its own or repair the damage incorrectly, the cell dies.
Figure 2b
Linkoping_Sweden
(https://2016.igem.org/Team:Linkoping_Sweden/Design）
Introduction: The problem they solve is the high cost of algal biofuels. They designed a structure that could control the metabolism of chlamydia. This construct is required to deliver inducible CRISPR/Cas9 knockdown that conveys specific metabolic target genes, directing carbon-derived cellular flux to triacylglycerol (TAG) synthesis.
Learning: Their pitcher plant demonstration illustrates the inducible function of the CRISPR/Cas9 system. Cas9 can be positively regulated by the light-induced LIP promoter. LIP can achieve the light-induced expression and target gene knockdown of Cas9. There are several variants of the inducible promoter that can be used to express genes such as Cas9 in response to a given stimulus. Cas9 acts as a knockout medium in the CRISPR/Cas9 system, requiring only very low Cas9 expression to achieve target gene knockdown. Finally, sgRNA acts as a binding protein gene, allowing rapamycin resistance to be used as a selective marker for successful gene knockdown.
Figure 2c
NTU-Singapore
(https://2016.igem.org/Team:NTU-Singapore/Description）
Introduction: Our project aims to improve the CRISPR/Cas9 toolkit for genome engineering and regulation. The Cas9/Cpf1 protein is widely adopted because of its simple programmability to generate targeted double-strand breaks. The cell's repair machinery will then repair the break, allowing us to edit the DNA. To date, several questions about this technology remain unanswered. First, despite the discovery of multiple Cas9 / Cpf1 proteins, we still do not know how efficiently each protein is compared to the other. In addition, the cutting efficiency of different cutting parts is also different. The popularity of the technology comes from its ability to increase knock rates. However, its efficiency still needs to be improved as it varies between target locations. The team's goal is to make a thorough comparison of different Cas9 proteins and improve the efficiency of their genome editing.
Learning: They used transcriptional activation domain fusion of dCas9 to activate a GFP reporter construct cotransfected into HEK293FT cells to determine the binding affinity of dCas9 to its target. The specificity of Cas9 can increase with the length of gRNA. So they screened out the best length of gRNA needed. Among other things, they used directed evolution to screen for more efficient SpCas9 variants in cleaved DNA to improve the efficiency of the system and improve the CRISPR/Cas9 system.
Figure 2d
UiOslo_Norway
（https://2018.igem.org/Team:UiOslo_Norway）
Introduction: Based on previous projects, the UiOslo team aims to develop a rapid detection kit for Candida albicans infection using CRISPR/dCas9. Vaginal samples were treated with glucanase to cleave yeast cell walls and expose fungal DNA. After that, a modified dCas9 enzyme fused with split β-lactamase was added. Using specially designed guide Rnas, the dCas9 complex binds adjacent to each other on Candida albica-specific DNA sequences. Activated β-lactamase cleaves its substrate nitrocifen, producing a colored product indicating the presence of Candida albicans.
Learning: Use the refined CRISPR-Cas9 system to detect specific DNA sequences in the Candida albicans genome by Cas9 endonuclide (dCas9) protein. The detection method using CRISPR-Cas9 is very convenient and can be productized at scale. Endonuclease activity was inhibited by inducing point mutations in the domain of conventional Cas9. Candida albican-specific, 20-nucleotide complementary guide Rnas were designed so that dCas9 binds only to the targeted DNA strand. Due to the specificity of CRISPR-Cas9, such a detection system has very little error. And by changing the guide RNA, the system can detect almost any DNA-containing organism with high accuracy.
Conculsion
Under the guidance of currently available solutions and regulations, we focused on the current epidemic situation, consulted relevant literature and proposed a preliminary design after team brainstorming, namely, to establish a nucleic acid detection method based on type ⅵ CRISPR⁃Cas system, and further improve the sensitivity of detection by introducing Csm6.
Dialogue with local aquatic enterprises
We visited Hainan Ruilai Aquaculture Co., LTD. The company is a leading enterprise focusing on the breeding of Penaeus vannamei larvae, and is an important standardized breeding base of Penaeus vannamei larvae in China. Through the field visit, we learned that Hainan Ruilai currently has three molecular biology testing laboratories, equipped with a complete set of molecular biology testing equipment, and adopts the national standard (or industry standard) method used by authorities to carry out daily pathogen monitoring on water sources, shrimps, biological bait, larvae and shrimps, etc. The experimental operation and control will be carried out by graduate students with theoretical and technical foundation of molecular biology. In the process of communication with the staff, we raised the question of "whether there are any microbial diseases in the process of breeding that cannot be controlled by the application of drugs", which attracted their attention. The staff said that such "difficult diseases" appeared in the process of breeding, and the use of drugs against specific pathogenic microorganisms still did not help. Finally, through further investigation, it was found that the root cause of this situation was the development of drug resistance of pathogenic microorganisms. Through our in-depth discussion, we found that the detection of microbial resistance has a significant role in aquaculture. At present, we are in a highly concerned about the health of human society, human health with the continuous development of the society will only become more and more the center of construction. Facing consumers with higher and higher requirements for the quality of food products, aquatic enterprises must respond to the call of The Times, conform to the needs of consumers, and invest a large number of resources into product quality control and improvement. "We need an accurate, convenient and rapid method for detecting microbial resistance," the farm technician stressed as we investigated the issue further.
These suggestions greatly expanded our thinking. In order to better determine the direction of the project, we began to investigate the current market pain points. In the process, we had in-depth communication with the following companies and improved our design iteratively based on their feedback.
In the project of experiment in the process, we found that at present most of the means of both need to be sequenced columns to can detect by Cas detection system, in order to activate the Cas nucleic acid enzyme activity detection signal, and RPA, LAMP isothermal amplification methods although the high sensitivity, but the amplification process prone to product pollution, causing false positive results. Especially for Cas14a, because it only recognizes ssDNA, in order to generate a single strand that can be recognized by Cas14a, a primer is modified with thiophosphate lipid during nucleic acid amplification to be tested. After adding T7 exonuclide, the unmodified strand will be degraded and only the target sequence to be tested will be retained. Therefore, Cas14a is less tolerant of the mismatch between crRNA and the nucleic acid to be tested. In addition, the nucleic acid amplification step will also affect the sensitivity and speed of detection because of the long reaction time.
Research on the market to understand the current problems facing aquaculture
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①Current context: The unique challenges facing aquaculture
In recent years, with the continuous expansion of aquaculture scale and the continuous improvement of the degree of intensification, 150 ~ 200 kinds of diseases can be monitored in 90 aquaculture varieties. Aquaculture diseases are characterized by wide epidemic areas, variety of diseases and high mortality. Aquaculture diseases cause annual economic losses of 40 billion to 50 billion yuan.
②Identification of problems: Field investigation of local aquaculture farms
We visited Hainan Ruilai Aquaculture Co., LTD. Through field investigation, we learned that Hainan Ruilai currently has three molecular biology testing laboratories, equipped with a complete set of molecular biology testing equipment, and adopts the national standard (or industry standard) method used by authorities to carry out daily pathogen monitoring for water sources, shrimps, biological bait, larvae, shrimp seedlings, etc. In the process of communication, we raised the question of "whether there are microbial diseases that cannot be controlled by the application of drugs in the process of breeding", which attracted their attention. The staff said that such "difficult diseases" appeared in the process of breeding and production, and the use of corresponding drugs for specific pathogenic microorganisms was still of no avail. Finally, through further investigation and testing, it was found that the root cause of this situation was the resistance of pathogenic microorganisms. After the conversation, the farm technicians put forward their demands -- "We urgently need an accurate, convenient and rapid microbial resistance detection scheme".
③Verifying requirements: interviewing various aquaculture farms
To verify the need for testing microbial resistance protocols on aquaculture farms in Hainan, we interviewed our key stakeholder, aquaculture farmers. Through field interviews, we learned the answers to three main questions.
1.What are the main problems in their breeding process?
2.Do they think microbial resistance testing will be needed in the market?
3.What characteristics do they want for microbial resistance testing methods?
Figure 3a
Hainan Ruilai Aquaculture Co., Ltd. is a leading enterprise focusing on the breeding of Penaeus vannamei and an important standardized breeding base of Penaeus vannamei. In order to meet the development needs of the company and cultivate a new generation of high-quality shrimp seedlings, in 2010, the company invested more than 20 million yuan in the sky blue water clear, the original ecological environment superior Mulan Bay to build a new 30 mu of seedling breeding base. The base has 16 shrimp production rooms, 320 breeding ponds. After the base was put into use, the breeding base with an annual production capacity of 4 billion shrimp seedlings greatly alleviated the contradiction between supply and demand of "Wangyi" shrimp seedlings. In 2012, the annual sales volume of "Wangyi" shrimp seedlings reached more than 10 billion. We know that hainan flexibly by field visits to currently has three testing laboratory of molecular biology, and equipped with a complete set of molecular biology detection equipment, using the authority to use national standard method of water daily, kiss, biological bait, larval shrimp, shrimp seedlings for specific pathogens such as monitoring, and by the theory and technology of molecular biology foundation student experiment operation and the control. When we talked with the staff of Ruilai Aquaculture Co., LTD., the antimicrobial resistance of pathogenic microorganisms in aquatic products we presented attracted their attention, and after in-depth discussion, they thought it was very necessary to detect microbial resistance. In response to our interview questions, the person in charge of them gave the following answers:
1.Microbial diseases of aquatic products occurred on a certain scale.
2.The emergence of some "intractable diseases" in aquaculture shows the need for microbial resistance testing.
3.As a tool to solve disease problems, the biggest requirement for it is high accuracy.
Figure 3b
Hainan Xiangtai Fishery Co., LTD., established in 2004, is a national agricultural industrialization key leading enterprise integrating seed and seedling cultivation, ecological breeding, feed production, aquatic product processing, food testing, storage and logistics, biotechnology and sales. The main products are tilapia, gold pomfret, shrimp and semi-finished premade dishes. Relying on the advantages of the whole industrial chain, we have established a perfect traceability system to realize the whole traceability of products from the source to the table. The company's products have passed the pollution-free product origin certification, and BAP, ASC, BRC, IFS, HACCP and other more than 10 international certification. In addition, Xiangtai Tilapia has also obtained Hainan Sea bream certification and "Hainan Tilapia" agricultural Geographical Indication Authorization. Nanxiangtai Fishery has been committed to the development and supply of global high-quality aquatic products, providing high-quality aquatic protein for the global human beings. In the future, the company said that it will continue to improve the ability of product research and development innovation, to become the national trust of aquatic products brand, to become a leader in the aquatic industry. In the conversation with the technical experts of Ruilai Aquaculture Co., LTD., we learned that microbial resistance is not only the concern of microbial diseases in aquaculture, but also the increasing focus of buyers. Therefore, in order to ensure product quality and build excellent brand reputation of pollution-free products, microbial resistance detection of aquatic products is an increasingly important part. In response to our interview questions, the person in charge of them gave the following answers:
1.The view that microbial resistance in aquatic products indirectly causes harm to human body has become a hot topic of consumer concern.
2.In today's health-oriented human society, microbial resistance detection has become an aspect that companies have to pay attention to and devote considerable energy to.
3.As for production and operation, the requirement for a detection method is to pursue convenience and speed on the basis of ensuring high accuracy.
Figure 3c
Hainan Zhongzheng Aquaculture Science and Technology Co., Ltd. is a standardized joint-stock company initiated by Hainan Tenglei Aquaculture Management Co., Ltd. is a scientific and technological enterprise integrating scientific research, development, production, sales and technical services, and is the leading enterprise of agricultural industrialization in Hainan province. In the industry, it was the first to pass ISO9001, GAP and other certifications, won the title of "Hainan Provincial Aquatic seed Farm", "Ministry of Agriculture Aquaculture healthy Aquaculture Demonstration Farm", won the 2015 China Penaeus Vannamei seedling "New enterprise" award, was named "2016 China Aquatic industry Top Ten healthy and safe seedling brand", Won the title of "Top Ten Seedling Enterprises of 2017", contracted to build the "Aquatic Animal Testing Center of Dongfang City, Hainan Province" to strictly control the quality and serve the surrounding customers. The company is located in Dongfang City, Hainan Province, where the water quality is excellent. It has established a seed and seedling breeding base with advanced facilities in Xinlong. With a fixed asset investment of more than 90 million yuan, the base now has more than 25,000 cubic meters of breeding water. It has advanced and scientific water treatment system, strict biological control system and perfect quality control system. It monitors the whole production process and strictly controls the quality to ensure that each batch of shrimp can reach SPF quality. In the process of talking with the person in charge of the company, we found that the company attaches great importance to the quality and safety of aquatic products. The officials said microbial resistance is a "stumbling block" in improving the quality of aquatic products. Therefore, in their view, it is very necessary to test microbial resistance.
response to our interview questions, the person in charge of them gave the following answers:
1.Quality control of aquatic products has become a huge challenge in the process of aquaculture
2.The detection of microbial resistance is an aspect of whether the quality of aquatic products is qualified, so there is a certain market demand for the detection of microbial resistance.
3.We urgently need a high accuracy, portable and fast microbial resistance detection device.
Figure 3d
Fujian Minwei Industrial Co., LTD is a modern fishery enterprise, national key leading enterprise and provincial leading enterprise in Marine industry. And yellow sea fisheries research institute of Chinese academy of fishery sciences, fujian fisheries research institute, Shanghai ocean university, jimei university and other research institutions actively carry out "production, study and research" project cooperation, surrounding seawater fish seed breeding and its related industry chain extension and so on many subjects for research, has made a number of scientific research, and scientific research achievements transformation. Set up a "national ocean fish zhangzhou comprehensive experimental station of industrial technology system", "Chinese rabbits - fujian fuding bass aquaculture professional technology exchange center", "fujian province academician experts workstation" "flower perch breeding in fujian province key laboratory", "fujian institute of aquaculture seawater fish test base", "fujian wei spent perch processing enterprises in fujian province engineering technology research center", such as scientific research Taiwan aims to help enterprises solve the technical problems encountered in the rearing, breeding and processing of seawater fish, develop new products, cultivate talents, improve the independent innovation ability of enterprises, research and develop innovative technological achievements, in order to obtain good social and economic benefits. The person in charge said that there is a lack of rapid and effective disease diagnosis technology on the market at present. We further learned through the interview that the professional ability of the staff of aquatic animal prevention and quarantine stations in many areas is poor, and the infrastructure needs to be improved, so it is still impossible to carry out rapid disease diagnosis. The breakthrough of practical diagnostic technology is an urgent problem to be solved in aquaculture industry.
In response to our interview questions, the person in charge of them gave the following answers:
1.Disease diagnosis technology is a major problem in the process of breeding.
2.Microbial resistance testing is a key aspect of the company, which has a strong market demand.
3.We need practical diagnostic technology and rapid and high precision microbial drug resistance detection devices.
Survey of local topography and location of Haikou
To better understand the local port information, as well as to find the appropriate river estuary for sample collection. We made a survey of the topography of Haikou.
Figure 4a.Topography of Haikou
For the topographic analysis of the whole island, we selected 53 of the estuaries and made a map.
Figure 4b. Terrain
Based on the terrain, whether the rivers are polluted by human actions in scenic spots, factories or other areas, we have selected 30 of them and will arrange to conduct seawater sampling at these 30 locations in turn around Hainan Province.
Figure 4c
Cooperation with microbiology related technology companies
01、Trends in the molecular diagnostics industry
Figure 4d
①Aging, increased incidence of infectious diseases and tumors
By 2030, there will be 360 million people over the age of 60. With aging, human immunity decreases, which leads to an increased risk of infectious diseases, tumors and other diseases, and the need for molecular diagnostics such as disease screening and tumor early screening increases.
②Urbanization and emerging viruses lead to epidemics of infectious diseases
By the end of 2021, the urbanization rate of permanent residents in China was 64.72%, and it is expected to reach 71.2% by 2050. Excessive urban population growth and more frequent contact with dense population mobility have provided favorable conditions for the spread and prevalence of infectious diseases. SARS in 2003, Ebola in 2014, Zika in 2016, COVID-19 in 2020 and the recent emergence of unexplained hepatitis in children have all posed new challenges to the global health system, as the accelerated rate of virus mutation makes it more difficult to control infectious diseases.
③The emergence of drug-resistant microorganisms poses a great threat
If effective measures are not taken, it is estimated that by 2050, 10 million people will die of drug-resistant bacteria worldwide every year, and the economic loss will reach 100 trillion US dollars. Microbial drug resistance has become a global public health crisis. Traditional bacterial culture requires a long detection time, and most viruses are difficult to or even cannot be cultured in vitro, while molecular diagnosis can rapidly diagnose microbial resistance. The Xpert® CarBA-R Assay carbapenem resistance gene test kit launched by Sapei in 2020. Five major carbapenem resistance genes blaKPC, blaNDM, blaVIM, blaIMP and BlaoxA-48 were also reported.
④The COVID-19 pandemic has accelerated the development of the industry, and PCR laboratories are springing up all over the country
Before the epidemic, PCR laboratories were mainly located in large Classⅲ Grade A hospitals, CDC, laboratories of scientific research institutes and other institutions, which had very high requirements on personnel quality, equipment and laboratory environment. Since the outbreak of the epidemic, nearly 10,000 PCR laboratories have been established in hospitals above the second level and third-party ICL, which greatly improved China's nucleic acid detection capacity in a short period of time and effectively shortened the "testing time for all". In the post-epidemic era, how to play the functions of these "costly" PCR laboratories has become a new topic. Molecular diagnosis in the hospital can not only solve the turn-around time (TAT) of detection, but also reduce the vacancy problem of PCR laboratories.
02、Product features
What we've designed is a portable test for antibiotic-resistant microorganisms in the ocean. The system is relatively small in size, can achieve rapid detection, human-computer interaction interface friendly and easy to operate. The system mainly has three modules, mainly divided into temperature regulation module, optical path detection module and Android screen display module. Functions that can be implemented (key benefits)
①Sample as you go
②Portability and miniaturization bring a wider range of application scenarios
Hierarchical diagnosis and treatment, some community outpatient clinics, secondary hospitals, township health centers, etc., also need molecular diagnosis, but these primary medical institutions lack professionals, and fewer medical personnel;
In emergency scenarios, the collection and delivery of clinical laboratory specimens takes a lot of time. How to minimize TAT has become a key factor restricting the rapid diagnosis and treatment of emergency medicine;
Border entry and exit quarantine, the increase of import and export goods increases the workload of border entry and exit quarantine, at the same time, the emergence of new viruses, increased the difficulty of quarantine, a little negligence will cause serious economic losses; In addition, airports, high-speed railway stations, ports and other places with dense personnel and large mobility, as well as field hospitals, all need portable, miniaturized, moderate flux and easy to operate diagnostic instruments.
Figure 4e
Figure 4f
During the visit to VIEWKER, HP team had a detailed discussion with Chief Engineer Yongjie Zhong, focusing on the hardware part of the marine microbial drug resistance testing instrument.
Q1:How to make the enzyme standard instrument more convenient
A1: flux, temperature control system (there must be heat dissipation, power supply and other power appliances in it, it limits its volume, only through a relatively small flux, as far as possible through a special method to limit its power can not be too large, there will also be sacrifices, such as similar to PCR to warm up and annealing must be high-powered components to meet, such as the traditional power does not need too large, because there are Power devices exist for the reason that the power supply limits its portability, if you want to portable must bring a power supply, then the instrument will not work for a long time
Q2: Is there a better way to detect fluorescence
A2: At present, microbial detection from the fluorescence detection method is only the plate method and MPN method, but these two methods have a disadvantage - it takes a long time and is cumbersome to use. And now the common method is mainly plate counting method. Compared with the MPN, the plate technology may be a little less effort, but it will cost more, you need to buy a lot of supplies and supporting some sealing machine and other things. This kind of fluorescence detection, and then want to be able to quantify, then the current words. There is no other method, plate technology. If you want to do the counting method, you can do it visually.
Q3: Is it necessary to detect nucleic acid accurately to a single base
A3: The current process of detection methods that require PCR amplification is very cumbersome and time-consuming, requiring multiple manual interventions
Test strips: sensitivity is very poor (nanodiamond is used to improve sensitivity, but the technology is not yet mature)
By single base non-amplification method can greatly simplify the process, and can be popularized, no biochemical related background personnel can also do (add samples and wait for the end of the test to collect data)
Q4：What are the current market instruments for marine microbial diagnosis, or nucleic acid detection instruments
A4: Most are done by PCR, for known microorganisms to know what type, based on genetic data to determine, or based on the characteristics of the plate colony, whether you use your eyes to count, or in the microscope. Under the microscope to count live bacteria, it is a method that belongs to this technique. And then there's a putative method, which is estimation. And then you can check the standard. Collectively, it's called the MPN method. Some of them are called esophageal method. Some are 51-well quantitative method, and some are 97-well quantitative method. And then the conventional method is the esophageal method. We dilute it in a gradient and then inoculate it. Inoculated into ten tubes, and then according to your tube. The long discoloration of that situation, and then to check the table. Finally, you can estimate a number of this microorganism.
Q5: Our project can be applied in what fields after
A5：Product quality control, water quality testing, pharmaceutical analysis, microbial fermentation, development of engineering bacteria, food safety, cosmetics and other production quality control (all qualitative OR quantitative testing of microorganisms / research OR factory)
Q6: What about the cost? About the cost of hardware, self-cleaning costs, durability and safety, etc.
A6: The hardware is about one or two hundred dollars, mainly the cost of the structure, which may be thousands; safety has to be verified through experiments, by manually going through the process and then integrated into the automated machine hardware. For each safety indicators to be considered clearly, sealing issues, material issues (corrosion resistance, permeability, etc.)
Q8: What are the existing instruments (in terms of nucleic acid detection - fluorescence-based detection)
A8: Enzyme marker, UV spectrophotometer, traditional impedance method, by indirectly detecting the current signal, but the stability is poor and the error is relatively large.
Q9: If the project is landed, how competitive can our project be in the market?
A9:The principle of drug resistance detection is based on colorimetric reaction and photoelectric detection, so the competitiveness is mainly reflected in the indicators of the embodied instruments. Now microbial detection methods are still based on the most traditional technology, only to be popularized by the integration of easy-to-operate instruments, which is of great importance to the whole industry.
03、Optimize the experimental scheme and achieve cooperation intention
Our products meet the development concept of "rapid detection" of VREWKR company, and compared with the traditional microplate reader, the cost is lower, more portable and the process is simpler, which has a broad market development space and a wide range of target users.
During the visit to the company, the head of the technical department discussed with our experimental group members, They introduced us to a synthetic nucleic acid analogue, peptide nucleic acid (PNA), consisting of nuclear bases aligned along a pseudopeptide skeleton, and suggested that we take advantage of PNA's ability to integrate the genetic information encoded by nucleic acids with the structural and functional properties of amino acids encoded by proteins. By specifically hybridizing with DNA or RNA to form stable PNA-DNA or PNA-RNA heterozygous double-stranded structure, it can exert its high biological stability and high affinity for binding to complementary nucleic acid sequences, so as to solve the problem of false positives in detection results. This feedback will promote us to carry out a new round of experimental scheme optimization and product upgrade. In the future, we will build a portable microbial resistance detector that can be mass-produced, low-cost and high-precision.
At present, Hainanu-China has reached a preliminary cooperation intention with Hainan Wekrypton Biotechnology Co., LTD., and we will complete the plans on the transformation of scientific and technological achievements, financing and instrument mass production in the future.
Ninth China Regional IGEM Exchange Conference
The team also participated in the 9th China Regional IGEM Communication Conference (CCIC), which invited several honorary guests, ranging from famous academicians to successful entrepreneurs, who gave valuable sharing on their understanding of the future of synthetic biology and related technical principles. At the meeting, we actively exchanged our project experiences with other teams and drew extensive guidance advice on hardware, and the CCiC Executive Committee Chair evaluated our project and provided us with useful suggestions to improve our presentation.
It is worth mentioning that we won the best hardware design award among the many teams in this CCIC!
Figure 4g |
Figure 4h |