Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 14(1): 1505, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36932065

RESUMO

Nucleic acid sensing powered by the sequence recognition of CRIPSR technologies has enabled major advancement toward rapid, accurate and deployable diagnostics. While exciting, there are still many challenges facing their practical implementation, such as the widespread need for a PAM sequence in the targeted nucleic acid, labile RNA inputs, and limited multiplexing. Here we report FACT (Functionalized Amplification CRISPR Tracing), a CRISPR-based nucleic acid barcoding technology compatible with Cas12a and Cas13a, enabling diagnostic outputs based on cis- and trans-cleavage from any sequence. Furthermore, we link the activation of CRISPR-Cas12a to the expression of proteins through a Reprogrammable PAIRing system (RePAIR). We then combine FACT and RePAIR to create FACTOR (FACT on RePAIR), a CRISPR-based diagnostic, that we use to detect infectious disease in an agricultural use case: honey bee viral infection. With high specificity and accuracy, we demonstrate the potential of FACTOR to be applied to the sensing of any nucleic acid of interest.


Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Animais , DNA/genética , Agricultura , Cabeça , RNA/genética , Sistemas CRISPR-Cas/genética , Técnicas de Amplificação de Ácido Nucleico
2.
J Vis Exp ; (184)2022 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-35781278

RESUMO

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider implications for the well-being of societies and economic stability. Recent years have seen several new isothermal diagnostic modalities emerge to meet the need for rapid, low-cost molecular diagnostics. We have contributed to this effort through the development and patient validation of toehold switch-based diagnostics, including diagnostics for the mosquito-borne Zika and chikungunya viruses, which provided performance comparable to gold-standard reverse transcription-quantitative polymerase chain reaction (RT-qPCR) based assays. These diagnostics are inexpensive to develop and manufacture, and they have the potential to provide diagnostic capacity to low-resource environments. Here the protocol provides all the steps necessary for the development of a switch-based assay for Zika virus detection. The article takes readers through the stepwise diagnostic development process. First, genomic sequences of Zika virus serve as inputs for the computational design of candidate switches using open-source software. Next, the assembly of the sensors for empirical screening with synthetic RNA sequences and optimization of diagnostic sensitivity is shown. Once complete, validation is performed with patient samples in parallel with RT-qPCR, and a purpose-built optical reader, PLUM. This work provides a technical roadmap to researchers for the development of low-cost toehold switch-based sensors for applications in human health, agriculture, and environmental monitoring.


Assuntos
Vírus Chikungunya , Infecção por Zika virus , Zika virus , Animais , Humanos , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Zika virus/genética , Infecção por Zika virus/diagnóstico
3.
Nat Biomed Eng ; 6(3): 246-256, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35256758

RESUMO

In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5% (95% confidence interval, 96.2-99.6%, 268 serum samples) and 98.5% (95% confidence interval, 91.7-100%, 65 serum samples) and approximately 2 aM and 5 fM (both concentrations are within clinically relevant ranges). The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings.


Assuntos
Febre de Chikungunya , Vírus Chikungunya , Dengue , Infecção por Zika virus , Zika virus , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/epidemiologia , Dengue/diagnóstico , Humanos , Zika virus/genética , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/epidemiologia
4.
Nat Commun ; 12(1): 724, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526784

RESUMO

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.


Assuntos
Técnicas Biossensoriais/métodos , Redes Reguladoras de Genes/genética , Glucose/análise , Ácidos Nucleicos/análise , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , Técnicas Biossensoriais/instrumentação , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/virologia , Glucose/metabolismo , Humanos , Ácidos Nucleicos/genética , Pandemias , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/fisiologia , Febre Tifoide/sangue , Febre Tifoide/diagnóstico , Febre Tifoide/microbiologia
5.
Nat Chem ; 12(1): 48-55, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31767994

RESUMO

The field of synthetic biology has used the engineered assembly of synthetic gene networks to create a wide range of functions in biological systems. To date, gene-circuit-based sensors have primarily used optical proteins (for example, fluorescent, colorimetric) as reporter outputs, which has limited the potential to measure multiple distinct signals. Here we present an electrochemical interface that permits expanded multiplexed reporting for cell-free gene-circuit-based sensors. We have engineered a scalable system of reporter enzymes that cleave specific DNA sequences in solution, which results in an electrochemical signal when these newly liberated strands are captured at the surface of a nanostructured microelectrode. We describe the development of this interface and show its utility using a ligand-inducible gene circuit and toehold switch-based sensors by demonstrating the detection of multiple antibiotic resistance genes in parallel. This technology has the potential to expand the field of synthetic biology by providing an interface for materials, hardware and software.


Assuntos
DNA de Cadeia Simples/química , Técnicas Eletroquímicas/métodos , Redes Reguladoras de Genes , Genes MDR , Alcanossulfonatos/química , Compostos Azo/química , Enzimas de Restrição do DNA/química , DNA de Cadeia Simples/genética , RNA Polimerases Dirigidas por DNA/química , Resistência a Múltiplos Medicamentos/genética , Técnicas Eletroquímicas/instrumentação , Fluoresceínas/química , Azul de Metileno/química , Microeletrodos , Hibridização de Ácido Nucleico , Estudo de Prova de Conceito , RNA Mensageiro/análise , Proteínas Virais/química
6.
Sci Rep ; 6: 27085, 2016 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-27255932

RESUMO

Tunnelling nanotubes and cytonemes function as highways for the transport of organelles, cytosolic and membrane-bound molecules, and pathogens between cells. During viral infection in the model organism Drosophila melanogaster, a systemic RNAi antiviral response is established presumably through the transport of a silencing signal from one cell to another via an unknown mechanism. Because of their role in cell-cell communication, we investigated whether nanotube-like structures could be a mediator of the silencing signal. Here, we describe for the first time in the context of a viral infection the presence of nanotube-like structures in different Drosophila cell types. These tubules, made of actin and tubulin, were associated with components of the RNAi machinery, including Argonaute 2, double-stranded RNA, and CG4572. Moreover, they were more abundant during viral, but not bacterial, infection. Super resolution structured illumination microscopy showed that Argonaute 2 and tubulin reside inside the tubules. We propose that nanotube-like structures are one of the mechanisms by which Argonaute 2, as part of the antiviral RNAi machinery, is transported between infected and non-infected cells to trigger systemic antiviral immunity in Drosophila.


Assuntos
Proteínas Argonautas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Organelas/metabolismo , RNA de Cadeia Dupla/genética , Proteínas Virais/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Animais , Proteínas Argonautas/metabolismo , Transporte Biológico , Comunicação Celular , Linhagem Celular , Dicistroviridae/genética , Dicistroviridae/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/microbiologia , Drosophila melanogaster/ultraestrutura , Drosophila melanogaster/virologia , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Organelas/microbiologia , Organelas/ultraestrutura , Organelas/virologia , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/crescimento & desenvolvimento , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
7.
Dev Comp Immunol ; 42(1): 85-92, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23684730

RESUMO

RNA interference (RNAi) controls gene expression in eukaryotic cells and thus, cellular homeostasis. In addition, in plants, nematodes and arthropods it is a central antiviral effector mechanism. Antiviral RNAi has been well described as a cell autonomous response, which is triggered by double-stranded RNA (dsRNA) molecules. This dsRNA is the precursor for the silencing of viral RNA in a sequence-specific manner. In plants, systemic antiviral immunity has been demonstrated, however much less is known in animals. Recently, some evidence for a systemic antiviral response in arthropods has come to light. Cell autonomous RNAi may not be sufficient to reach an efficient antiviral response, and the organism might rely on the spread and uptake of an RNAi signal of unknown origin. In this review, we offer a perspective on how RNAi-mediated antiviral immunity could confer systemic protection in insects and we propose directions for future research to understand the mechanism of RNAi-immune signal sorting, spreading and amplification.


Assuntos
Infecções por Arbovirus/imunologia , Arbovírus/imunologia , Vesículas Revestidas por Clatrina/metabolismo , Drosophila melanogaster/imunologia , Interferência de RNA , Animais , Antígenos Virais/imunologia , Proteínas Argonautas/metabolismo , Vesículas Revestidas por Clatrina/imunologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/virologia , Endocitose/imunologia , Humanos , Imunidade Inata/genética , RNA Helicases/metabolismo , Interferência de RNA/imunologia , RNA de Cadeia Dupla/imunologia , RNA Viral/imunologia , Ribonuclease III/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...