Poster: Leiden

Welcome to the poster of iGEM Leiden 2020! Click on the different sections to read more about it.


Team members
Violette Defourt, Tom Langelaar, Tijn Delzenne, Kelly van Strien, Amber Schonk, Aukje Beers, Eugene Golov, Marijn van den Brink, Sebastian Tandar, Tim van den Akker, Sinisha Jovikj, Lucy Chong, Güniz Özer Bergman and Joey Meijdam.

PIs: Han de Winde, Dennis Claessen.
Supervisors: Marjolein Crooijmans, Floor Stel, Jonah Anderson, Laurens ter Haar, Maarten Lubbers.
Advisors: Chiel van Amstel, Daniël Tan, Tobias Fecker, Jo-Anne Verschoor, Daan van Tol, Charlotte de Ceuninck, Carli Koster.


Rapidemic: A novel modular point-of-care diagnostic tool for rapid epidemic response

This year’s COVID-19 outbreak demonstrated how the world is impacted by a pandemic, causing over one million deaths worldwide and severely damaging the quality of life of billions. Rapid diagnostics are vital to keep an outbreak under control and reduce the need for disrupting measures. Here, we present an innovative, modular technique called Rapidemic that allows for the rapid detection of nucleic acids of pathogenic species in a future outbreak. By combining targeted amplification (RPA), nickase-based GQ DNAzyme generation (LSDA), and DNAzyme-catalyzed oxidation, our method can reliably and rapidly detect DNA or RNA and provides the user with a simple colorimetric output. Because it does not require a lab or external power source, our technology enables point-of-care testing in both high- and low-resource areas. This way, Rapidemic offers a global solution to a global problem and allows us to be one step ahead in tackling Disease X!

Color-blind friendly

Maybe you have noticed how the colors of our poster changed once you clicked on this little section on the top right. This is how people with the most common form of color blindness, deuteranopia, would see our poster. As you can see, everything is still clearly readable! Click once more on the section on the poster to remove this effect.*

Color-blind people can experience issues when reading scientific figures as they are not able to distinguish certain colors. This makes certain heat maps, fluorescence microscopy images and graphs impossible to interpret (Fig. 1). Therefore, we made sure our figures, wiki and poster are color-blind friendly, so that color-blind people are also able to understand everything. In addition, the colorimetric reaction of our technology is also visible for color-blind people, ensuring that everyone can interpret our kit. Lastly, we have provided a color blindness guide, giving the tools to other iGEM teams to make their wiki colorblind-proof and contribute to a more inclusive community.

Fig. 1 | The reds and greens on a heatmap are indistinguishable for color-blind people. The image on the left shows a seemingly clear readable heatmap for people without color deficiencies the heatmaps. However, when we apply a color blindness filter, the two colors become indistinguishable from each other, making it almost impossible for color-blind people to interpret this figure. (Figures adapted from:

*Unfortunately, this effect does not work in Safari and Internet Explorer. Please use a different browser, like Chrome, to see this effect.

Challenge and Solution

Infectious diseases travel faster than ever in our increasingly globalized world, as demonstrated by the recent outbreak of coronavirus disease 2019 (COVID-19) 1.

While a rapid diagnostic response at the start of an outbreak is crucial for containing the spread, this strategy failed due to the lack of rapid, accurate, and accessible tests 2,3.

PCR is a versatile and accurate test but cannot be used in point-of-care settings. In contrast, existing point-of-care tests often lack sufficient sensitivity and their development is long 4-7.

Thus, there is an urgent need for accessible tests that can be quickly adapted to new pathogens.

We developed a rapidly adaptable detection technology, called Rapidemic, that is based on nucleic acid amplification, like PCR. However, its ability to be applied in point-of-care settings makes the test accessible and allows a quick epidemic response.

Challenge image

Diagnostic testing is important to 'flatten the curve'. Frequent testing enables the detection and subsequent isolation of infectious people. This strategy effectively reduces the number of infections and postpones the peak in number of infected people.

Our technology

Our technology consists of three consecutive well-characterized reactions that allow for the detection of a target pathogen.

1. Amplification of target sequence

During the first reaction, Recombinase Polymerase Amplification (RPA), a pathogen-specific sequence is copied at an exponential rate using special primers that contain an overhang. In contrast to PCR, which relies on thermocycling, RPA occurs at a constant temperature.8-10.

2. Production of DNA reporter

This is followed by the Linear Strand-Displacement (LSDA) reaction during which a nickase initiates a single-stranded cut at a site created by the primer overhang. The GQ section after the cut is subsequently amplified to detach this single-stranded DNA-based reporter or DNAzyme.

3. Visible color change

In the final reaction, this reporter associates with potassium ions and hemin to form a three-dimensional structure that can oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB) 11. Upon oxidation, a product is produced that provides a clear color change that is visible to the naked eye.

Our technology

Rapidemic is based on nucleic acid amplification, a highly sensitive method compared to antigen detection 12-14. To circumvent a time-consuming protein translation step for detection, we chose a DNA enzyme (DNAzyme) as reporter.

Rapidemic's specific set of primers allows rapid adaptation to a multitude of pathogens.

To avoid the need for laboratory equipment, we combined a colorimetric readout with isothermal DNA amplification. Such an amplification method is performed at a low and constant temperature. The point-of-care and low-cost technology make the test accessible to anyone.



The generic Rapidemic test kits can be purchased and distributed worldwide in anticipation of an outbreak.


Our kit comes into play when a new outbreak happens.


The pathogen is immediately screened and its genome is published to make it accessible internationally. Similar to PCR, primers specific for a pathogen are designed to identify the infected individuals. The same primer sequence can be used to locally produce primers for the Rapidemic test kits. In doing so, the strain on large international companies that provide the reagents is reduced in a crisis situation.


The primers can be added to the generic kits.


Afterwards, the kits must be tested and validated.


The tests are then distributed, after which trained technicians can perform the tests massively in crowded areas, such as in airports, schools, elderly homes. Alternatively, the tests could also be useful in low-resource areas, where the need for laboratory-independent diagnostic tools is more important.

Proof of Concept

The three biomolecular reactions were serially coupled to detect the presence of yeast genome (Fig. 1).

  • Yeast was used as a safer alternative to other pathogens.
  • Dithiothreitol (DTT) present in the first reaction was found to inhibit subsequent color reaction.

We solved this by using a combination of two strategies (Fig. 1).

Strategy 1 - Optimizing Dilution Scheme
We adjusted the amounts of product from each preceding reaction that were used in each subsequent reactions.

Strategy 2 - Inactivating DTT
We attenuated the reducing power of DTT by lowering the pH of the oxidation buffer.

The color change was finally enabled by the use of pH 3.8 phosphate-citrate buffer and an optimized dilution scheme: a 10-fold dilution from RPA to LSDA and an 80-fold dilution from LSDA to the color reaction (Fig. 2).


Fig. 1 | Our technology integrates three biochemical reactions in a serial manner. The product of each preceding reaction was used to start subsequent reactions. Such handling strategy also caused the product from each preceding reaction to be diluted in the following reaction(s). The inhibition of the oxidation reaction by DTT was attenuated by optimizing the dilution scheme and the pH of the oxidation buffer.

Figure 2

Fig. 2 | DTT inactivation and dilution allowed color to be generated during the oxidation reaction. a) A higher color generation could be achieved at lower buffer pH, confirming the inactivation of DTT. b) A lower amount of unpurified RPA products is required to allow color generation. c) Visual comparison of color generation using purified/unpurified RPA products at different dilution rates. Values represent the dilution rate used between RPA and LSDA as well as LSDA and TMB oxidation, respectively.

Reaction Modeling

Non-specific amplification during RPA gave rise to false-positive signal.

Dimethyl sulfoxide (DMSO) was used in our amplification reactions to minimize the impact of non-specific RPA amplification.

DMSO concentration has to be optimal:

  • Too less – false-positive signal persists
  • Too much – signal from the target is lost

What our model does
Our model predicts reaction progression, given reaction conditions as input.

Example simulation

Without DMSO, non-specific amplification in RPA gets carried over through subsequent reaction, resulting in a false-positive report at the final signal readout (TMB oxidation).


Fig. 1 | Simulated reaction progress of RPA, LSDA, and TMB oxidation in the absence of DMSO. Starting template concentration was set to 500 (relative fluorescence unit), and incubation time of RPA, LSDA, and TMB oxidation to 20, 40, and 20 minutes, respectively. RFU; relative fluorescence unit. A650; absorbance at 650 nm.

How the model is used to optimize DMSO concentration

The model was used to predict a DMSO concentration that maximizes the endpoint signal difference between the positive and negative tests (dA650).

1.33% DMSO was found to be the best in maximizing dA650!

 Predicted endpoint absorbance signal intensity at different DMSO concentrations.

Fig. 2 | Predicted endpoint absorbance signal intensity at different DMSO concentrations. Starting template concentration was set to 500 (relative fluorescence unit), and incubation time of RPA, LSDA, and TMB oxidation to 20, 40, and 20 minutes, respectively. The difference between endpoint positive and negative signals was calculated as the arithmetic difference between the two.


Our prototype shows how the technology holds the potential to be further optimized into a rapidly adaptable point-of-care diagnostic tool.

  • Design
    The design of the prototype consisted of 3x3 reactions to enable subsequent amplification and detection of (1) a test sample, (2) a negative control and (3) a positive control (Fig. 1).
  • Use
    The prototype can be used by adding the test sample to the well of the first reaction (RPA), followed by incubation and subsequent transfer to the LSDA and oxidation reactions.
  • Duration
    Through the window on the top, the color change could be observed by the naked eye after 70 minutes (20 minutes after the start of the third reaction).
  • Costs
    The costs of the reagents were estimated to be $ 1.45 per reaction, which is similar to low-cost commercial amplification kits (Fig. 2).

Fig. 1 | Scheme of the prototype design. a) First Rapidemic prototype made from black cardboard. b) Snapshots of three oxidation reactions with LSDA product (sample), phosphate citrate buffer (- control) and synthetic DNAzyme (+ control). Time shown beside each snapshot represented the time elapsed after the start of the TMB oxidation reaction.

Fig. 2 | Cost estimation of our technology. The estimated cost of reagents per- 20 µL was compared to that of commercial RT-PCR, LAMP and RPA assays.


We have identified a potential use-case for our kit: screening potentially infected individuals during influenza A outbreaks. We found a number of potential end-users, including clinicians, schools, and nursing homes. Finally, we have spoken to numerous potential partners, identified multiple funding sources, and filed a national patent application.

In the coming year, we would like to:

  • carry out experiments to optimize Rapidemic into a fast, accurate, and fully functional pathogen detection method.
  • ensure that our intellectual property is protected by filing an international patent application.
  • simultaneously start the design process of our kit in collaboration with the innovation agency IDE Group.
  • itemize the needs of potential end-users and use these to develop a prototype. This prototype will be tested in the lab and in a clinical setting.
  • in later stages establish agreements with manufacturers, distributors, and NGOs of the project to bring Rapidemic one step closer to the end-users.
Human Practices - Science

At the start of our project, we reached out to the 2017 iGEM Mantis project 15, to understand the challenges they faced and to identify our starting point. Our team decided to opt for a cell-free system, comprised of enzymes and rather stable nucleic acids 16. Based on opinions of experts, we opted for the RPA amplification method 17, which is sensitive and can be used equipment-free. For simple result interpretation, we decided to try out the GQ-DNAzyme/hemin oxidation 18 and integrate controls.

Human Practices - Application

Identifying a niche in the field of infectious diseases required us to balance the science of our kit with other factors, such as the medical need for better diagnostics, patients’ preferences, the gap in the diagnostics market, but also the feasibility of the implementation. We first thought about focussing on specific diseases, such as Lyme disease 19, Cholera 20, tuberculosis 21 or urinary infections 22. However, in doing so, Rapidemic would be in direct competition with the sensitivity, cost, quality, and accuracy of the current diagnostic methods for these diseases. Subsequently, we looked into mosquito-borne diseases that affect millions globally each year 23. Although there is a high demand for better diagnosis, these diseases are not transmitted through direct contact, and therefore do not require RDTs. Eventually, experts told us to take advantage of the modularity of our kit, and focus on the group of respiratory RNA viruses, that have great epidemic potential 24.

Human Practices - Regulation

Understanding the regulations that govern the market of diagnostic devices globally is important. Experts helped us understand what it takes to bring a kit to a market where the standards are extremely high, versus a market where low(er) cost applications are often the norm 25. We quickly understood that the validation would lead to an inevitable delay in the commercialization of the kits. However, such a delay could be reduced by using pre-produced generic kits, which are completed locally with specific primers 26. Additionally, continuously adapting and testing the kits of specific RNA viruses can help in building up a reputation and ensure quality 27.

Human Practices - User

From an accessibility viewpoint, self-testing RDTs are optimal as they do not require medical assistance and can be distributed and sold in pharmacies, directly to the population. We interviewed patients from around the world, entrepreneurs and doctors that have worked in areas with limited lab or hospital access to understand whether self-testing is the most appropriate option 28. The input gathered indicates that patients as well as doctors and developers think it is best to entrust trained staff with test kits, as patients are not eager to perform minor medical interventions and the quality of the result should be safeguarded. With this option, the tests could be used easily in crowded areas while ensuring their proper use and disposal. Additionally, patients and doctors shared with us that the design should match the use of the kits, and robustness and minimal handling are crucial 29.

Human Practices - Ethics & Culture

Doing business in African countries is certainly not the same as doing business in Europe. Topics like culture, social taboos, trust and poverty play a significant role in determining if our product is going to be a success or a failure. Social considerations should not be forgotten when trying to enter the African market. Gaining trust is of vital importance. The best strategy regarding the quickest entry into the market is licensing our product to a company with a good reputation and which is trusted by the local population 30.

Human Practices - Affordability

Diagnostics should be sold at a very low price as usually they are government-funded, or funded by foundations or NGOs 31. Therefore, businesses have to work with an immuable, limited budget. An economical test would allow governments to be able to test more, and repeatedly. Molecular tests tend to more economical 32 which was confirmed in our experiments, where the cost of a single reaction was $1.45. This could further be lowered if mass-produced. To ensure equal access to diagnosis, we came up with a hybrid income model where the price of the tests would depend on the income in the countries. High income countries, would then pay a "premium" price that would co-fund the purchase of tests by NGOs or governments in lower income countries 33.

Human Practices - Logistics

For transport, experts told us that robustness and temperature insensitivity are necessary for distribution in lower-income countries 34, where refrigerated transport over long distances is difficult and extremely costly. Robustness and simplicity would safeguard the quality but also reduce the cost. Therefore, we looked into freeze-drying the reagents, and into paper-based and other compact, simple designs for Rapidemic.

Human Practices - Disposal

Through contact with the diagnostic companies, we have quickly understood that RDT developers do not integrate the end-of-life considerations, such as environmental or (health) safety, into the developmental stages of the products 35. Developing a medical product for low-resource countries with inadequate infrastructure can present certain risks, which should be taken into account during the design process 36. After our kit has been used, it will be classified as hospital waste and should therefore be treated as such.

There is a common tendency regarding waste disposal across the world. If not landfilled, everything is often incinerated. One way to decrease the impact of the kits would be to use recycled content to reduce the unnecessary incineration of virgin material. Additionally, if the kits are incinerated, all the potentially infectious material, which should already be lysed, is killed. Instead, if the hospital waste is placed in a landfill to which the population has access, the content of the kits could present a risk. The potentially infectious content should therefore already neutralized and the kits should be sealed after used.

Lastly, our team visited the Netherlands' largest waste plant, Zavin, where we had the opportunity to ask about the current medical waste disposal practices and received a guided tour of the facilities.

To finance our research, learn more from experienced partners, and gain other valuable support we approached companies to help us with our project. We want to put the main sponsors in the spotlights.

Itility is a leader in the field of data science. This combined with their social responsibility and knowledge made them decide to support iGEM. They believe in the power of data science combined with synthetic biology.

IDE Group focuses on projects that can change lives in a meaningful way, such as a reliable rapid diagnostic test platform for HIV or COVID-19. They achieve this with their development, manufacturing and entrepreneurial skills.

Medical Delta is an interdisciplinary collaboration composed of over 280 scientists of academic hospitals and universities of applied sciences that work together with companies and governments to find technological solutions for problems that arise in health care.

The CHDR is an independent institute that specializes in cutting-edge early-stage clinical drug research. CHDR supports initiatives of motivated and curious students, such as our project. “We believe that it is every scientist’s responsibility to help educate student researchers and professionals.”

And thanks to all our other partners and sponsors!


We added a new part to the iGEM Registry of Standard Biological Parts and improved an existing part.

  • The new part BBa_K3343000 is a guanine-quadruplex DNAzyme with highest peroxidase-mimicking activity reported in literature (2016) 37. We determined its molar maximum substrate conversion rate (kcat) and substrate affinity (Km), and we confirmed its peroxidase-mimicking activity in a range of buffers of different pH.
  • The improved part BBa_K3343001 is a modified version of the most widely used guanine-quadruplex DNAzyme 37. We improved the existing part BBa_K1614007 by adding an adenine residue to the 3' end of its ssDNA sequence to increase its activity and tolerance to lower pH. In doing so, the DNAzyme became applicable for Rapidemic's low-pH reaction conditions.

Future Prospects

We have uploaded a pre-print of our results on bioRxiv. However, we would still like to improve several aspects of our technique:

  1. False-positives
    Our first priority is to reduce the false-positive rate of our amplification method to achieve high specificity. In the coming months, we will redesign our primer sequences to prevent off-target interactions (primer dimers) from occurring.
  2. One-pot reaction
    We aim to optimize the reaction conditions to combine RPA, LSDA and oxidation to reduce the number of handling steps. Furthermore, we need to design a lysis buffer to extract viral RNA from samples. Finally, we would like to explore alternative methods to stabilize the proteins in the RPA pre-mix, as the current stabilizing agent DTT interferes with the oxidation reaction.
  3. Hardware
    We aim to design a hardware prototype of our kit. From literature and expert interviews, we have learned that our device could be made using microfluidics-based or paper-based technology. Furthermore, we determined that we should create a minimum viable product (MVP). In the coming year, we will present such an MVP to potential end-users.
References and Acknowledgements

References from literature

[1] Kawachi, I. et al. Globalization and health. Oxford University Press (2007).
[2] Kelly-Cirino, C. D. et al. Importance of diagnostics in epidemic and pandemic preparedness. BMJ Glob. Heal. 4, e001179 (2019).
[3] Natesan, S. et al. Ramping up of SARS CoV-2 testing for the diagnosis of COVID-19 to better manage the next phase of pandemic and reduce the mortality in India. VirusDisease (2020). doi:10.1007/s13337-020-00622-x
[4] Bruning, A. H. L. et al. Rapid tests for influenza, respiratory syncytial virus, and other respiratory viruses: A systematic review and meta-Analysis. Clin. Infect. Dis. 65, 1026–1032 (2017).
[5] Tang, Y. W. & Stratton, C. W. Advanced techniques in diagnostic microbiology. in Advanced Techniques in Diagnostic Microbiology (eds. Tang, Y. W. & Stratton, C. W.) 31-51 (2006). doi:10.1007/0-387-32892-0
[6] Keipp Talbot, H. & Falsey, A. R. The diagnosis of viral respiratory disease in older adults. Clinical Infectious Diseases 50, 747–751 (2010).
[7] Peeling, R. W., Murtagh, M. & Olliaro, P. L. Epidemic preparedness: why is there a need to accelerate the development of diagnostics? The Lancet Infectious Diseases 19, e172–e178 (2019).
[8] Mullis, K. B. & Faloona, F. A. Specific Synthesis of DNA in Vitro via a Polymerase-Catalyzed Chain Reaction. Methods Enzymol. 155, 335–350 (1987).
[9] Corman, V. M. et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance 25, 2000045 (2020).
[10] Piepenburg, O., Williams, C. H., Stemple, D. L. & Armes, N. A. DNA detection using recombination proteins. PLoS Biol. 4, 1115–1121 (2006).
[11] Travascio, P., Li, Y., Sen, D. DNA-enhanced peroxidase activity of a DNA aptamer-hemin complex. Chemistry & Biology 5, 505-517 (1998).
[12] Bruning, A. H. L. et al. Rapid tests for influenza, respiratory syncytial virus, and other respiratory viruses: A systematic review and meta-Analysis. Infect. Dis. 65, 1026–1032 (2017).
[13] Tang, Y. W. & Stratton, C. W. Advanced techniques in diagnostic microbiology. in Advanced Techniques in Diagnostic Microbiology (eds. Tang, Y. W. & Stratton, C. W.) 31-51 (2006). doi:10.1007/0-387-32892-0
[14] Keipp Talbot, H. & Falsey, A. R. The diagnosis of viral respiratory disease in older adults. Clinical Infectious Diseases 50, 747–751 (2010).
[37] Li, W. et al. (2016). Insight into G-quadruplex-hemin DNAzyme/RNAzyme:adjacent adenine as the intramolecular species forremarkable enhancement of enzymatic activity. Nucleic Acids Research: 44(15), p. 7373-7384.


We would like to thank all the people and instances that have helped us with our Human Practices:
[15] iGEM Wageningen Mantis 2017
[16] Prof. dr. Janneke van de Wijgert, Niek Savelkoul iGEM Mantis 2017
[17] iGEM EPFL 2019 ViTest, Dr Armand Paauw & Dr Hans van Leeuwen
[18] Dr Thomas Caltagirone
[19] Dr Hein Sprong, RIVM
[20] René Paulussen, Mondial Diagnostics
[21] Dr Heleen Koudijs
[22] Dr Lieselotte Hardy, ITM
[23] Dengue/TB/Malaria patients, dr Helene van Oortschot
[24] Dr Pim Martens, Dr Armand Paauw, Dr Hans van Leeuwen
[25] Qserve, Erik van Vught, Ward Heij, Monitor Deloitte
[26] Dr Otto Kroesen
[27] René Paulussen, Modial Diagnostics, Guus Eskens, CARE NL
[28] Dr Heleen Koudijs, Dr Sophie van Baalen, Rathenau Instituut
[29] John Tonkinson, DCN DX, René Paulussen, Mondial Diagnostics, Dr Aldrik Velders and Vittorio Saggiomo
[30] Dr Otto Kroesen, Dr Heleen Koudijs
[31] John Tonkinson, DCN DX, Niek Savelkoel, iGEM Mantis 2017, Dr Dam
[32] Prof Dr Janneke van de Wijgert
[33] Dr Sophie van Baalen, Rathenau Instituut
[34] Guus Eskens, CARE NL, Heleen Koudijs
[35] Dr Assica Permata, Dr Heleen Koudijs
[36] Bram de Graaf, NNRD, Ron Roffel, Zavin, Gerard Vincent, Rad Bag Solutions