The widespread transmission and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) call for rapid nucleic acid diagnostics that are easy to use outside of centralized clinical laboratories. Here we report the development and performance benchmarking of Cas13-based nucleic acid assays leveraging lyophilised reagents and fast sample inactivation at ambient temperature. The assays, which we named SHINEv.2 (for 'streamlined highlighting of infections to navigate epidemics, version 2'), simplify the previously reported RNA-extraction-free SHINEv.1 technology by eliminating heating steps and the need for cold storage of the reagents. SHINEv.2 detected SARS-CoV-2 in nasopharyngeal samples with 90.5% sensitivity and 100% specificity (benchmarked against the reverse transcription quantitative polymerase chain reaction) in less than 90 min, using lateral-flow technology and incubation in a heat block at 37 °C. SHINEv.2 also allows for the visual discrimination of the Alpha, Beta, Gamma, Delta and Omicron SARS-CoV-2 variants, and can be run without performance losses by using body heat. Accurate, easy-to-use and equipment-free nucleic acid assays could facilitate wider testing for SARS-CoV-2 and other pathogens in point-of-care and at-home settings.
Design of nucleic acid-based viral diagnostics typically follows heuristic rules and, to contend with viral variation, focuses on a genome’s conserved regions. A design process could, instead, directly optimize diagnostic effectiveness using a learned model of sensitivity for targets and their variants. Toward that goal, we screen 19,209 diagnostic–target pairs, concentrated on CRISPR-based diagnostics, and train a deep neural network to accurately predict diagnostic readout. We join this model with combinatorial optimization to maximize sensitivity over the full spectrum of a virus’s genomic variation. We introduce Activity-informed Design with All-inclusive Patrolling of Targets (ADAPT), a system for automated design, and use it to design diagnostics for 1,933 vertebrate-infecting viral species within 2 hours for most species and within 24 hours for all but three. We experimentally show that ADAPT’s designs are sensitive and specific to the lineage level and permit lower limits of detection, across a virus’s variation, than the outputs of standard design techniques. Our strategy could facilitate a proactive resource of assays for detecting pathogens.
Harnessing genomic data and predictive models will provide activity-informed diagnostic assays for thousands of viruses and offer rapid design for novel ones. Here we develop and extensively validate new algorithms that design nucleic acid assays having maximal predicted detection activity over a virus’s full genomic diversity with stringent specificity. Focusing on CRISPR-Cas13a detection, we test a library of ~ 19,000 guide-target pairs and construct a convolutional neural network that predicts Cas13a detection activity better than other techniques. We link our methods by building ADAPT, an end-to-end system that automatically leverages the latest viral genome data. We designed optimal species-specific assays for the 1,933 vertebrate-infecting viral species within 2 hours for most species and 24 hours for all but 3. ADAPT’s designs are sensitive and specific down to the lineage-level for the range of taxa we tested, including ones that pose challenges involving genomic diversity and specificity. They also exhibit significantly higher fluorescence and lower limits of detection, across a virus’s full spectrum of genomic diversity, than designs from standard techniques. ADAPT is available in an accessible software package and can be applied to other detection technologies to enhance critically-needed viral diagnostic and surveillance efforts.
The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.
Engineers design for an inherently uncertain world. In the early stages of design processes, they commonly account for such uncertainty either by manually choosing a specific worst-case and multiplying uncertain parameters with safety factors, or by using Monte Carlo simulations to estimate the probabilistic boundaries in which their design is feasible. The safety factors of this first practice are determined by industry and organizational standards, providing a limited account of uncertainty; the second practice is time intensive, requiring the development of separate testing infrastructure. In theory, robust optimization provides an alternative, allowing set based conceptualizations of uncertainty to be represented during model development as optimizable design parameters. How these theoretical benefits translate to design practice has not previously been studied. In this work, we analyzed present use of geometric programs as design models in the aerospace industry to determine the current state-of-the-art, then conducted a human-subjects experiment to investigate how various mathematical representations of uncertainty affect design space exploration. We found that robust optimization led to far more efficient explorations of possible designs with only small differences in an experimental participant's understanding of their model. Specifically, the Pareto frontier of a typical participant using robust optimization left less performance “on the table” across various levels of risk than the very best frontiers of participants using industry-standard practices.
Engineers design for an inherently uncertain world. In the early stages of design processes, they commonly account for such uncertainty either by manually choosing a specific worst-case and multiplying uncertain parameters with safety factors or by using Monte Carlo simulations to estimate the probabilistic boundaries in which their design is feasible. The safety factors of this first practice are determined by industry and organizational standards, providing a limited account of uncertainty; the second practice is time intensive, requiring the development of separate testing infrastructure. In theory, robust optimization provides an alternative, allowing set based conceptualizations of uncertainty to be represented during model development as optimizable design parameters. How these theoretical benefits translate to design practice has not previously been studied. In this work, we analyzed present use of geometric programs as design models in the aerospace industry to determine the current state-of-the-art, then conducted a human-subjects experiment to investigate how various mathematical representations of uncertainty affect design space exploration. We found that robust optimization led to far more efficient explorations of possible designs with only small differences in an experimental participant’s understanding of their model. Specifically, the Pareto frontier of a typical participant using robust optimization left less performance “on the table” across various levels of risk than the very best frontiers of participants using industry-standard practices.
When you use a computer it also uses you, and in that relationship forms a new entity of melded agencies, a â ȂIJcentaurâ Ȃİ inseparably human and nonhuman. Networks of interaction in an organization similarly form â ȂIJorganizational centaursâ Ȃİ , melding humans, technologies, and organizations into an inseparable sociomateriality. By developing a convex optimization toolkit for conceptual engineering we sought to shape these centaurs. How do organizations go from a high-level concept (â ȂIJletâ Ȃ Źs make an airplaneâ Ȃİ ) to a â ȂIJdesignâ Ȃİ , and in that process what blurred lines between humans and computers bring opportunities for research? We present three metaphors that have been useful lenses across our field sites: considering design models as maps shows how centaurs apportioned legitimacy; looking at design models as mirrors illuminates how they sought validation in their perspectives; and treating design models as participants recognizes their opinions and agency as equivalent to other entities in these centaurs.
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