RESUMO
Coronavirus disease 2019 (COVID-19) has become pandemic since March 11, 2020. Thus, development and integration in clinics of fast and sensitive diagnostic tools are essential. The aim of the study is a development and evaluation of a one-step quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay (COVID-19 Amp) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection with an armored positive control and internal controls constructed from synthetic MS2-phage-based RNA particles. The COVID-19 Amp assay limit of detection was 103 copies/ml, the analytical specificity was 100%. A total of 109 biological samples were examined using COVID-19 Amp and World Health Organization (WHO)-based assay. Discordance in nine samples was observed (negative by the WHO-based assay) and discordant samples were retested as positive according to the results obtained from the Vector-PCRrv-2019-nCoV-RG assay. The developed COVID-19 Amp assay has high sensitivity and specificity, includes virus particles-based controls, provides the direct definition of the SARS-CoV-2 RdRp gene partial sequence, and is suitable for any hospital and laboratory equipped for RT-qPCR.
Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Testes Diagnósticos de Rotina , Feminino , Genoma Viral/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Adulto JovemRESUMO
Theranostic approaches rely on simultaneous diagnostic of a disease and its therapy. Here, we designed a DNA nanodevice, which can simultaneously report the presence of a specific RNA target through an increase in fluorescence and cleave it. High selectivity of RNA target recognition under near physiological conditions was achieved. The proposed approach can become a basis for the design of DNA nanomachines and robots for diagnostics and therapy of viral infections, cancer, and genetic disorders.
Assuntos
DNA Catalítico/genética , Neoplasias/genética , RNA/química , RNA/metabolismo , Viroses/diagnóstico , DNA Catalítico/química , DNA Catalítico/metabolismo , Fluorescência , Humanos , Neoplasias/química , Nanomedicina TeranósticaRESUMO
Nucleic acids play a central role in all domains of life, either as genetic blueprints or as regulators of various biochemical pathways. The chemical makeup of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), generally represented by a sequence of four monomers, also provides precise instructions for folding and higher-order assembly of these biopolymers that, in turn, dictate biological functions. The sequence-based specific 3D structures of nucleic acids led to the development of the directed evolution of oligonucleotides, SELEX (systematic evolution of ligands by exponential enrichment), against a chosen target molecule. Among the variety of functions, selected oligonucleotides named aptamers also allow targeting of cell-specific receptors with antibody-like precision and can deliver functional RNAs without a transfection agent. The advancements in the field of customizable nucleic acid nanoparticles (NANPs) opened avenues for the design of nanoassemblies utilizing aptamers for triggering or blocking cell signaling pathways or using aptamer-receptor combinations to activate therapeutic functionalities. A recent selection of fluorescent aptamers enables real-time tracking of NANP formation and interactions. The aptamers are anticipated to contribute to the future development of technologies, enabling an efficient assembly of functional NANPs in mammalian cells or in vivo. These research topics are of top importance for the field of therapeutic nucleic acid nanotechnology with the promises to scale up mass production of NANPs suitable for biomedical applications, to control the intracellular organization of biological materials to enhance the efficiency of biochemical pathways, and to enhance the therapeutic potential of NANP-based therapeutics while minimizing undesired side effects and toxicities.
Assuntos
Aptâmeros de Nucleotídeos , Nanomedicina/métodos , Nanopartículas , Técnica de Seleção de Aptâmeros/métodos , Animais , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Imunoterapia , Camundongos , Nanopartículas/química , Nanopartículas/uso terapêutico , Neoplasias/terapia , Ácidos Nucleicos/química , Ácidos Nucleicos/uso terapêuticoRESUMO
Synthetic molecular machines have been explored to manipulate matter at the molecular level. Here, we designed a multifunctional DNA nano-construct, dubbed a 'DNA minimachine' (DMM), which (i) tightly binds complementary DNA; (ii) recognizes specific fragments with high selectivity and (iii) amplifies output signals. DMM1 detects lower concentrations of both single-stranded DNA and double-stranded DNA compared to a conventional probe. This study sets a direction towards the development of molecular machines for selective, sensitive and cost-efficient DNA analysis.
