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1.
Nat Biomed Eng ; 2022 May 30.
Article in English | MEDLINE | ID: covidwho-1873504

ABSTRACT

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.

2.
Viruses ; 13(8)2021 08 13.
Article in English | MEDLINE | ID: covidwho-1376992

ABSTRACT

While investigating a signal of adaptive evolution in humans at the gene LARGE, we encountered an intriguing finding by Dr. Stefan Kunz that the gene plays a critical role in Lassa virus binding and entry. This led us to pursue field work to test our hypothesis that natural selection acting on LARGE-detected in the Yoruba population of Nigeria-conferred resistance to Lassa Fever in some West African populations. As we delved further, we conjectured that the "emerging" nature of recently discovered diseases like Lassa fever is related to a newfound capacity for detection, rather than a novel viral presence, and that humans have in fact been exposed to the viruses that cause such diseases for much longer than previously suspected. Dr. Stefan Kunz's critical efforts not only laid the groundwork for this discovery, but also inspired and catalyzed a series of events that birthed Sentinel, an ambitious and large-scale pandemic prevention effort in West Africa. Sentinel aims to detect and characterize deadly pathogens before they spread across the globe, through implementation of its three fundamental pillars: Detect, Connect, and Empower. More specifically, Sentinel is designed to detect known and novel infections rapidly, connect and share information in real time to identify emerging threats, and empower the public health community to improve pandemic preparedness and response anywhere in the world. We are proud to dedicate this work to Stefan Kunz, and eagerly invite new collaborators, experts, and others to join us in our efforts.


Subject(s)
Disaster Planning , Lassa Fever/epidemiology , Lassa virus/physiology , Africa, Western/epidemiology , Disaster Planning/methods , Humans , Lassa Fever/genetics , Lassa Fever/prevention & control , Lassa Fever/virology , Lassa virus/genetics , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/immunology , Nigeria/epidemiology , Pandemics , Polymorphism, Genetic , Receptors, Virus/genetics , Receptors, Virus/immunology
3.
Nature ; 582(7811): 277-282, 2020 06.
Article in English | MEDLINE | ID: covidwho-980299

ABSTRACT

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.


Subject(s)
CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems/genetics , Microfluidic Analytical Techniques/methods , Virus Diseases/diagnosis , Virus Diseases/virology , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Drug Resistance, Viral/genetics , Genome, Viral/genetics , HIV/classification , HIV/genetics , HIV/isolation & purification , Humans , Influenza A virus/classification , Influenza A virus/genetics , Influenza A virus/isolation & purification , Microfluidic Analytical Techniques/instrumentation , RNA, Guide/genetics , SARS-CoV-2 , Sensitivity and Specificity
4.
Nat Commun ; 11(1): 5921, 2020 11 20.
Article in English | MEDLINE | ID: covidwho-939436

ABSTRACT

The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), a sensitive and specific diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We identify the optimal conditions to allow RPA-based amplification and Cas13-based detection to occur in a single step, simplifying assay preparation and reducing run-time. We improve HUDSON to rapidly inactivate viruses in nasopharyngeal swabs and saliva in 10 min. SHINE's results can be visualized with an in-tube fluorescent readout - reducing contamination risk as amplification reaction tubes remain sealed - and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-qPCR with a sample-to-answer time of 50 min. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.


Subject(s)
Betacoronavirus/isolation & purification , CRISPR-Associated Proteins/metabolism , Molecular Diagnostic Techniques/methods , Biological Assay , COVID-19 , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Fluorescence , Humans , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , SARS-CoV-2
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