The world has still been impacted by the COVID-19 pandemic since 2019. The coronavirus has deprived millions of lives and over 230 million people have been infected, which are huge challenges to the health system. Faced with the severe pandemic and the emergence of constantly mutated viruses, most countries have taken measures to test the virus and identify infected people as the primary basis of all actions. However, the widely employed fluorescent polymerase chain reaction (PCR) is still difficult to be practically used in some underdeveloped countries, which delays the general prevention and pandemic control. Therefore, in this project, we designed a visual virus detection platform based on synthetic biology to enable cheap but efficient virus detection in the areas with limited resources.
Build on the past iGEM successes
To build our detection platform, from both a technical perspective and a human practice level, we relied on the projects developed in previous years by other iGEM teams, such as:
2019 DUT_China A
The achievements of the previous biological research in virus detection are the important fundamental supports to our project. Therefore, we hope that we can help future iGEM teams build their detection programs and advance this area through cooperation and communication.
Since we want to implement our tests in the real world, we need to understand the real challenges confronted by us. Therefore, we firstly interviewed front-line workers engaged in virus detection. Secondly, we designed a questionnaire and launched a survey to understand the public awareness of our project and synthetic biology. Finally, we interacted with other iGEM teams and received suggestions for the improvement of our project. On this page, you can see how the feedback of this survey helped us improve our projects!
To learn more about current nucleic acid test approaches, we interviewed two front-line medical technicians. Through the communication and consultation with the them, we gained a lot of knowledge and valuable suggestions to improve our projects.
① Expert: Yajuan Chang
Deputy Chief Inspector, Department of Clinical Laboratory, the First Affiliated Hospital of Heilongjiang University of Chinese Medicine
Research direction: Clinical laboratory diagnostics
Could you tell us what steps are required from sample collection to the completion of detection, and what are the environmental requirements of these operations?
The way of the specimen collection in the clinical collection department should be professionally and correctly performed. The quality of specimens affects whether the subsequent results can meet the qualification control or not. We start from the reagent preparation in a laboratory, and then enter the detection area to collect the samples. We have different sampling tubes to carry depending on the situations. If the sampling tubes contain inactivating reagents, the samples do not need to be inactivated and can be directly dropped into the tubes. If the tubes do not have the inactivating reagent, the samples need to be inactivated first prior to the test experiment. Then, it is the step of the nucleic acid extraction and amplification. Finally, we read and interpret the test results. The whole process takes about 3.5-4 hours.
What do you think is the most difficult part of nucleic acid test, or what is needed to be improved?
Now, the nucleic acid detection technology has become quite mature. At the beginning, the time of the amplification reaction needed 2 hours and 50 minutes, but now it only takes 40-60 minutes to complete. In addition, the management was chaotic at the beginning, and there are peak hours for people to come in for the test. Now, these situations have been very much improved.
Through the communication with Mr. Chang, we know that the current testing process is complicated and the sample needs to be inactivated. Therefore, we designed the flow of the platform to treat the samples first with a protease and created hardware to reduce any potential biohazard risk.
②Expert: Mr. Zhu
Frontline nucleic acid testing medical staff
Why do we need to detect the output of PCR products in real time? We can observe the results after the complete PCR, isn't that okay?
First of all, we are doing high-throughput analyses with 96 samples at a time using the fluorescence quantitative PCR machine. In addition, the fluorescence quantitative PCR is more sensitive than the traditional ones. We can also use it to learn other details, such as virus copy numbers.
So, the virus copy number varies from person to person?
It does. Even for the same infected person, it can vary at different sampling times. Usually, within the first week of infection, the amount of virus is very high, and as time goes on, it decreases. Therefore, we have certain requirements for the routine sampling. Sampling of the infected population in the first few days after the infection, it is much easier to detect the virus than the situations at the latter stages during the medical treatment. So, there is a timeline of detection. If someone has the symptom of infection, we continuously take sampled from him or her to monitor the changes.
What you have described are the common testing methods. How will the nucleic acid test be conducted in the places where laboratory is unavailable?
A rapid detection technology has emerged, but it also needs a device. The instrument is small, but may only detect one or two samples each time. After the sample is put into the instrument, it takes about 30-40 minutes to get nucleic acid test result, while the fluorescent quantitative PCR method may take one and half hours. Nevertheless, the quick method is not widely used, and does not guarantee a high accuracy.
Through the communication with Mr. Zhu, we understood the deficiencies in the current nucleic acid testing process and the reasons accounting for them. We find that there is an urgent need for detection techniques with at least comparable sensitivity and efficiency as the currently used methods. Thus, we tested the threshold of copy numbers (Experiment). At the same time, we did not want to only focus on the test speed by sacrificing its accuracy.
After talking to the experts, we reconsidered the design and applications of our project. We wanted to know if people would really like our platform, so we launched online questionnaires to evaluate the public opinions to our approach. We think it is important to collect inputs and opinions from the end users, the society.
Population sample: 540 questionnaire I and 521 questionnaire II Based on statistical analyses, we can see that the responses to our surveys came from people with different backgrounds or occupations (Figure 1), and located all over China (Figure 2，3).
Figure 1. Proportion of the survey respondents by occupations.
Figure 2. Proportion of survey respondents by location
Figure 3. Regional distribution of the respondents.
Based on the collected data, we found that only 16% of the public had never taken any nucleic acid test for COVID-19. A detailed investigation was conducted among the 455 people who had taken nucleic acid test. The results indicated that the nucleic acid test was free to the public in nearly half of the regions in China, and the cost in most regions was less than 100 CNY (Figure 4).
Figure 4. Statistics of nucleic acid test costs.
For the waiting time, most people could receive their test results within 1 to 3 days, and very few people waited for 10 or more days. Clearly, there is still room for the improvement in the detection efficiency (Figure 5).
Figure 5. Waiting time for nucleic acid results in China
Knowledge about detection: According to the feedbacks to our questionnaires, only 1/4 of the respondents knew the principle of nucleic acid detection (Figure. 6). Therefore, we focused on the popularization of basic knowledge of nucleic acid detection in our media publicity.
Figure 6. The extent of the understanding of existing nucleic acid detection methods among the respondents of the surveys.
Recognition and feedback to our technology:
We discovered the public thoughts about our project in another questionnaire. After analyzing the public responses to our testing process, we found that approximately 70% of the participants supported this method. Yet, a small portion of the participants held skeptical or neutral attitude on it. We got an average score of 3.96 out of 5 points (Figure 7). To raise people's awareness of the convenient and quick test method, we also promoted a series of propagandas, which is displayed in our [Education and Public Engagement]section.
Figure 7. Public recognition of detection methods with visible readout.
Finally, we inquired the public for their opinions regarding the part that was most needed to be optimized in our test (Figure 8). The success key to any novel detection method is the amplification of virus signal. Thus, we analyzed the flowchart (Figure 8) to understand what the public thought to improve our project. Based on the chart, the detection efficiency accounted for the most expectation of about 40%, followed by the operation difficulty and overall cost. The lowest one was the “need to optimize”. Our optimized design to improve the detection efficiency is presented in the [Results] section.
Figure 8. The parts that need improvement based on the public opinions.
Critical feedback: In the questionnaire, we received valuable suggestions from an anonymous respondent in Shanghai, which made us realize that our design needed to be improved based on the practical circumstances. For example, for the first suggestion of this respondent, we designed a zinc finger protein [Design] that was not affected by RNase. The third and fourth suggestions from this person also provided insightful inputs to our project. The following is the feedback received form this person:
- It should be considered that after leaving the laboratory, RNase-free environment cannot be achieved, and there are a large number of RNase in saliva. Therefore, you need to consider whether the viral RNA will be quickly degraded after peeling or sampling, which may be the main source of false negative results when using this test method.
- You should carefully confirm the specificity of the primers used in the amplification. Whether the primers may somehow match the DNA or RNA of other microbes or oral epithelial cells. In addition, the basal G4 levels of the extracted RNA and reverse transcription products may also create background color, which may be the main sources of false positive results in this detection method.
- To prevent the potential situation of the false negative mentioned above, it is suggested to evaluate the levels of the RNAs from the oral epithelial cells. This may ensure you that the sampled RNAs are not quickly degraded.
- To prevent the potential situation of false positive mentioned above, it is suggested to create precise physical and chemical environment during the detection based on the pH and ion requirements of the G4 formation. This may reduce the possible G4 formation by other sequences. Meanwhile, you should always include a negative control (a stable linear DNA) and a positive control (a stable G4 structure-containing DNA).
Team communication and cooperation
Through the ICII (Into China Into iGEM) platform, we cooperated with many Chinese universities to promote the iGEM events and team projects, and produced joint promotional posters. At the same time, we also got to know a number of interesting topics. After extensive communication, we provided many suggestions to each other, which was beneficial to the promotion of the projects. (Learn more details about this, please check in the [Collaboration] section)
Communication and cooperation with the team CPU_CHINA
In August 2021, through the interaction with the team CPU_CHINA, we found that both sides had their designs based on the CRISPR system and related proteins. Therefore, we shared experience in the experiments of protein purification and extensively discussed the applications of sgRNA and dsDNA. We mutually provided suggestions to the related experiments. (CPU_Collabration)
During the CCiC's online Q&A session in September 2021, we discussed protein assembly experiments with the CPU team. In our experiments, we encountered the problem that the combination of strains BW and T1 could not successfully express the nCas9-linker-Phi29 fusion protein under our tested induction conditions. After communicating with the CPU team, we realized that E. coli system could not well express proteins with molecular weights over 200 kDa, although this is feasible in an eukaryotic chassis, such as yeast. It means that the expression of our fusion protein in bacteria was too large and complex. Therefore, the members of the CPU team introduced the methods used by them to purify the SpyCatcher and SpyTag in their project, which could relieve the burden of bacterial protein expression. They suggested us to test this system for the assembly of our proteins in vitro.
On September 5, 2021, after analyzing the data, we designed the expression of nCas9 and Phi29 proteins separately based on their suggestions, and attached the two proteins by two short peptides slig and SH3, respectively, followed by the assembly of the associated proteins through the interaction between the two peptides. We finally succeeded in obtaining two components, nCas9-slig and Phi29-SH3, and tested their assembly in vitro.