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Demonstrate
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With our modular approach, we prototype all units of our diagnostic device, and integrate them into a customizable platform. We use cell-free synthetic biology to distinguish between pathogens with our universal detection cascade.
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Sample processing
We chose to combine heat lysis and isothermal amplification (RPA) to extract our target RNA from patient samples.
- We used RPA to amplify DNA from heat lysed E. coli.
- We conducted RPA and transcription from an in vitro DNA on paper.
- We amplified and transcribed an in vitro DNA target to RNA concentrations detectable by our readout circuit
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We validated the three necessary modules of the sample processing (cell lysis, DNA amplification and transcription) with a rapid and sensitive method, RPA.
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Readout circuit
We chose Cas13a for pathogen identification because of its specificity for nucleic acid sequence detection.
- We characterized Cas13a and its detection limit with native and lyophilized protein, with in vitro and in vivo sources of RNA, in bulk and on paper.
- We designed fluorescent and colorimetric readouts, and used a synthetic aptamer as a detection tool.
- We tested targets from common pathogens and showed the orthogonality of virus detection versus bacterial detection.
We proved the robustness and universality of our Cas13a-based fluorescence readout circuit.
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Readout circuit
We chose a disposable paper strip combined with a reusable fluorescence detector to analyse our samples.
- We optimized the paper support for Cas13a functionality.
- We built the most sensitive and cheapest fluorescence detector ever created by an iGEM team to our knowledge.
- We successfully used our fluorescence detector to detect Cas13a activity on paper.
We created a detection chip that is portable, functional and affordable, for the distribution of our diagnosis device, CascAID.
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