Sexually transmitted infections (STIs) remain a significant public health concern worldwide, with limited access to rapid, reliable, and user-friendly diagnostics contributing to delayed treatment and ongoing transmission. Current testing methods often require laboratory infrastructure, trained personnel, and multiple steps, creating barriers for widespread use.
TRACE reimagines STI diagnostics through synthetic biology by combining rapid, room-temperature nucleic acid amplification using recombinase polymerase amplification (RPA) with a highly specific detection platform that produces a simple colorimetric readout. To optimize specificity, cost and test durability, we investigated two detection strategies: the CRISPR-Cas12a system and our novelly applied DNA hybridization-based method for STI detection. Detection is sequence specific and the system can easily be extended to additional pathogens as well as re-used in completely different applications.
Our team worked across Wet Lab, Dry Lab, and Human Practices, achieving the following (See more under Results):
Through this interdisciplinary approach, we designed a diagnostic platform for STIs that is data-driven, supported by modelling and shaped by society.
One million. That’s the number of people getting newly infected with sexually transmitted infections (STIs) worldwide every day, according to the World Health Organization [1]. These infections are primarily transmitted through sexual contact and pose a serious and growing global health challenge.
The most common STIs include chlamydia, gonorrhea, syphilis, trichomoniasis, and human papillomavirus (HPV). If left untreated, they can cause severe health problems such as infertility, chronic pelvic pain, complications during pregnancy, and even certain cancers. Furthermore, some STIs increase the risk of HIV transmission [2]. For more information see our page Education page on STIs.
Many STIs remain undiagnosed because early infections are often asymptomatic. Combined with persistent social stigma, this leads to limited testing, fewer conversations, and ongoing transmission.[3] Moreover, medical care for STIs is primarily focused on prevention, such as vaccination, barrier protection, and treatment. However, sexual protection does not completely eliminate the risk of infection, and many people are unaware of this. Regular screening for STIs enables earlier treatment and should become a routine part of healthcare. The problem is not simply a lack of awareness. Current testing options are not designed for regular screening. In Switzerland, as in other countries, getting tested for STIs usually requires visiting a doctor, going to a pharmacy, or sending a sample to a laboratory. Self tests exist, but most still require mailing the sample and waiting several days for the result. This process is often expensive, time-consuming, and not very private, leading to an even greater number of undiagnosed cases. As part of Human Practices we ran a survey to understand the main reasons why people avoid testing and did interviews to understand what risks are associated with this. (See more under Contribution: Human Practices)
There is therefore a clear need for a rapid, affordable, and private diagnostic tool that people can use by themselves, anytime and anywhere. Stakeholders and our survey highlighted the need for this.
TRACE aims to close this gap with a multiplexed, freeze-dried, paper-based, cell-free at home diagnostic test capable of detecting five of the most prevalent STIs worldwide: Human papillomavirus (HPV16 & HPV18), Gonorrhea (Neisseria gonorrhoeae), Chlamydia (Chlamydia trachomatis), Syphilis (Treponema pallidum) and Trichomoniasis (Trichomonas vaginalis). For more information on each pathogen, check out our Product Page, an excellent example of integrated Human Practices.
With TRACE, we aim to provide a test that is low-cost, rapid, portable, and user-friendly. Our vision is to transform STI testing into a routine and make it easy to buy and use, just like a pregnancy test (Check out our Entrepreneurship Page). By designing a multiplexed platform that supports multiple sample types, we ensure detection even when infections are localized to specific anatomical zones. Guided by our entrepreneurship analysis, we estimate a cost of approximately $25 for end-users ($15 in retail stores), with each test delivering results in under one hour.
According to our epidemiological model, implementing our proposed test could substantially contribute to the WHO’s goal, helping reduce STI cases from 374 million in 2020 to fewer than 150 million annually by 2030.
From the beginning, our team was drawn to diagnostics because it aligned with our vision of creating a project that could be integrated into society. When our team member Tatia first suggested developing a test for STIs, some of us felt hesitant due to personal discomfort around the topic. This very discomfort came from the weight of social unacceptability, the feeling that it was a subject we weren’t supposed to address, reserved only for those considered to have crossed a line and thus tested positive. We then recognized that this very stigma is a barrier to healthcare and decided to tackle it head-on. After every team meeting, interviews with experts and conversations with friends about our project, we became increasingly committed to this important topic and, soon, instead of being afraid of opening up conversations about it, we couldn’t stop bringing it up. Because that is how you break a taboo, by talking about it until it becomes a normality. Just as it should be. Ultimately, this project changed us from within and we hope it will make a similar impact outwardly.
Our approach was inspired by a scientific paper describing a cell-free diagnostic system for the Zika virus that combined RPA for amplification with CRISPR for detection [4]. We presented our initial plan to adapt this paper’s methods for STI detection to ETH Professor Sven Panke, who confirmed the feasibility of our idea.
Our experimental workflow was structured to evaluate and validate each functional component of the system in isolation before integrating them into a complete assay (Check out our Engineering and Cycles Page). This modular approach ensured that we could systematically identify and address potential sources of error or inefficiency at each stage. Similarly, our Dry Lab models were designed in a modular fashion, allowing them to be validated independently and to serve as building blocks for more complex models (see our page Overview). To organize our project, we divided it into three main components: the amplification method, for which we chose RPA, and two detection methods to be tested, namely CRISPR and DNA hybridization (For more information on this see our Notebook Page and our Experiments & Protocols Page). Diagnostics with the LbCas12a protein, which is a popular approach based on CRISPR-Cas technology, is known for its high sensitivity and specificity [8]. DNA Hybridization, on the other hand, is a novel approach in this context that offers potential benefits such as lower cost, simplicity and stability. [9] We confirmed its feasibility in consultation with PhD Cheng-Han Yang. In short, we retained CRISPR as a safety net, as there exists extensive literature about this topic, in case DNA hybridization, a less commonly used technique, did not perform as expected.
Our first focus was the amplification step, because without a reliable way to multiply even tiny amounts of DNA, no detection method could succeed. RPA offered exactly what we needed: a fast method compatible with minimal equipment and low incubation temperatures, that is well-suited for use in portable or field-deployable diagnostic assays and point-of-care applications [7]. It utilizes a combination of recombinase enzymes, single-stranded DNA-binding proteins (SSBs), and strand-displacing polymerases to facilitate primer binding and DNA synthesis. [6]. As a first step, we designed and validated RPA primers, and performed RPA at different temperatures and incubation times and with varying initial DNA template concentrations to assess amplification efficiency. Notably, our computational model predicted amplification behavior in the same order of magnitude as the experimental results, demonstrating that the parameters of our RPA model capture the system dynamics within a realistic and acceptable range. See more on our: Reaction Model Page. In addition, we implemented both symmetrical [10] and asymmetrical RPA [11], since DNA hybridization requires single-stranded products, while LbCas12a is activated by double-stranded targets to produce fluorescence.
Following this, we evaluated the two separate detection strategies: one based on LbCas12a-mediated trans-cleavage activity, and the other based on DNA hybridization using labeled probes. By testing each detection method separately, we confirmed that both were able to produce fluorescence signals using RPA-amplified DNA as input. We tested whether these methods remained functional under different experimental conditions by varying factors such as reaction time, temperature, and reagent concentrations. In parallel, we ran exploratory “RPA-on-a-paper” trials to probe an end-to-end paper workflow.
As a final step, we tested our detection systems using commercially available lateral flow strips that are compatible with FAM- and biotin-labeled samples. Both the LbCas12a-based and the DNA hybridization-based methods were applied in this format to evaluate whether they produce clear and reliable signals on paper. This allowed us to demonstrate that our detection reactions can be transferred onto a simple and user-friendly format, as well as moving closer, step by step, to a functional prototype of our diagnostic tool.
For aspects that we could not easily test in the lab, such as the shelf life of the test or how the liquid behaves on the strip, the dry lab supported us by creating models and running simulations. This provided us with useful insights and helped us better understand these elements without relying solely on physical experiments.
Our fluorescent tests confirmed that our DNA-hybridization chemistry was working effectively, showing that our probes could specifically recognize target pathogen sequences. However, a home diagnostic test cannot rely on lab equipment such as fluorescence readers, which are impractical for at-home use. To create a simple, user-friendly, and easily interpretable signal, we turned to gold nanoparticles.
On a lateral flow strip, these nanoparticles are conjugated to our mobile DNA probes. When the probes bind to the target pathogen DNA in the sample, the complex flows along the strip until it is captured at a specific test line. It is a double-binding strategy, where both a mobile probe and a fixed capture probe are required, that ensures high specificity and reduces false positives, creating a visible band that can be read without any instruments.[12] Unlike traditional approaches, this design combines DNA hybridization with a cell-free, paper-based diagnostic platform, making it a novel and robust method for multiplexed STI detection. This approach also aligns with broader global health goals by enabling affordable, accessible, and low-resource STI diagnostics (Check out our Sustainability Page).
Due to time constraints, we were not able to fully implement this experiment before the wiki freeze, but the experimental plan has been designed and will be executed. Check out our Outlook Page.
The DNA-hybridization method proved not only successful but also the most suitable approach for diagnostics, offering advantages in stability, cost, and reliability. In contrast, our integrated CRISPR/Cas12a–RPA model revealed a critical bottleneck: at low DNA concentrations, Cas12a cleaves target DNA faster than RPA can amplify it, creating a “deadlock” that prevents a detectable signal.
This in-silico insight uncovered a fundamental design flaw before we did the experiments, saving time and resources. While the model suggested a workaround of temporally separating RPA and Cas12a reactions to allow sufficient amplicon accumulation, the simplicity and robustness of DNA-hybridization make it the most practical choice for our diagnostic platform.
Learn how we incorporated feedback from stakeholders and experts on the Integrated Human Practices Page and discover our strategies for real-world deployment on the Entrepreneurship Page.