In November of 2019, after the NYUAD iGEM group returned from the giant jamboree in Boston, a conservation research scientist from the University of Freiburg, Dr. Johannes Penner, reached out to the team and proposed a problem for the group to address given our diagnostic history. After outlining the current issues with diagnostic methods, Dr. Penner indicated that a field use device similar to our past solutions, giving results within 20-30 minutes or so would be a real game-changer in monitoring amphibian populations for both fungi and allowing conservationists, local agencies, etc. to act immediately.
Following the lead by Dr. Penner, we sought to understand the amphibian decline, the epidemiology of the fungal disease, and the amphibians themselves. The unstructured and branched-out research into the topics helped the team better refine our approach to the problem in later stages.
Producing the ultimate diagnostics device necessitated looking at every option for the components. To do so, we imagined the whole testing pathway beginning with sample collection and ending with a successful receipt of the result.
Following in-depth research of point of care diagnostics and the fungal disease, the team identified a clear pathway towards developing the product. After identification of the technical variables, the biology, engineering, and user-mapping team went through a rigorous research and engagement phase to ensure the solution was practical and scalable.
Mapping users and exploring the why behind a diagnostic device necessitated an understanding of where diagnosis falls in infectious disease control. We divided our user mapping process into three stages based on differences in disease dynamics and appropriate management actions: Pre-arrival, Invasion front, Epidemic/Established. We used these fronts to help us understand where a diagnostic device falls into the animal infectious disease biosecurity process.User Mapping call_made
The standard method for sample collection is a swab on the skin of the animal. Recently, eDNA — testing the habitat instead of the animal itself - has proven to be effective and we're developing solutions for that as well in the later stage of our project. After gathering this mix of genetic material, the next step in a diagnostic product is to extract and purify the DNA. For that, we're looking at: chemical methods such as lysis buffers with simultaneous purification using silica matrices or paramagnetic particles (PMPs) and mechanical methods such as bead beating and sonication. For purification, magnetic bead extraction, silica matrices, and Whatman FTA lysis paper are options we're trialing. The options we're trialing are marked with green lines on the figures.Sample Collection call_made Nucleic Acid Extraction call_made
Amplification and Detection
We then use an ultra-sensitive and high-throughput nucleic acid detection technique. An amplification reaction, like RPA and LAMP, will be coupled with our detection system, like CRISPR/Cas12a and CRISPR/Cas13, to sense our target gene.
Next in the pathway is the reaction medium where the amplification and detection methods discussed above happen. We're looking at options ranging from PCR tubes with modifications such as pierceable membranes, mini- opentrons, magnet operated and paramagnetic particles based systems, microfluidic chips, paper-based biosensors, and sliding cartridges to complete integrated solutions in literature such as MAD-NAAT and LabOnAChip.Case Studies call_made Sample Prep/ Reaction Medium Pathways call_made
Reporting and Sensing
Finally, for reporting and sensing the results of the test, we looked at multiple methods, and are experimenting with fluorescence and lateral flow assays. For sensing these reporting mechanisms, we are prototyping and testing various kinds of portable fluorescence detection mechanisms, and creating machine learning algorithms to bring quantification to lateral flow assays. The options we are trialing are marked green on miroReporting Mechanism call_made Measurement call_made
Fungal disease outbreak analysis increasingly occupies an informatic space where the development of toolkits that facilitate rapid analysis and dissemination of diverse data types are central to effective disease management. That is why the NYUAD iGEM Group is also developing a comprehensive database to retrieve and manage diagnostics data and a web API that can integrate with any surveillance system.Data and Infectious Diseases call_made