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1.
Nat Protoc ; 15(11): 3663-3677, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33005038

RESUMO

The complexity of current nucleic acid isolation methods limits their use outside of the modern laboratory environment. Here, we describe a fast and affordable method to purify nucleic acids from animal, plant, viral and microbial samples using a cellulose-based dipstick. Nucleic acids can be purified by dipping in-house-made dipsticks into just three solutions: the extract (to bind the nucleic acids), a wash buffer (to remove impurities) and the amplification reaction (to elute the nucleic acids). The speed and simplicity of this method make it ideally suited for molecular applications, both within and outside the laboratory, including limited-resource settings such as remote field sites and teaching institutions. Detailed instructions for how to easily manufacture large numbers of dipsticks in house are provided. Using the instructions, readers can create more than 200 dipsticks in <30 min and perform dipstick-based nucleic acid purifications in 30 s.


Assuntos
Celulose/química , Ácidos Nucleicos/isolamento & purificação , Animais , Bactérias/química , Humanos , Técnicas de Amplificação de Ácido Nucleico/economia , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Plantas/química , Fatores de Tempo , Vírus/química
2.
Nat Commun ; 11(1): 4774, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963224

RESUMO

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL-1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.


Assuntos
Líquidos Corporais/química , Técnicas de Diagnóstico Molecular/métodos , Ácidos Nucleicos/isolamento & purificação , Sepse/diagnóstico , Candida/genética , Candida/isolamento & purificação , Candidíase/diagnóstico , Contagem de Colônia Microbiana , Doenças Transmissíveis/diagnóstico , DNA/isolamento & purificação , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Reação em Cadeia da Polimerase/métodos , RNA/isolamento & purificação , Sensibilidade e Especificidade , Sepse/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Urina
3.
Biosens Bioelectron ; 169: 112592, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32942143

RESUMO

Global health and food security constantly face the challenge of emerging human and plant diseases caused by bacteria, viruses, fungi, and other pathogens. Disease outbreaks such as SARS, MERS, Swine Flu, Ebola, and COVID-19 (on-going) have caused suffering, death, and economic losses worldwide. To prevent the spread of disease and protect human populations, rapid point-of-care (POC) molecular diagnosis of human and plant diseases play an increasingly crucial role. Nucleic acid-based molecular diagnosis reveals valuable information at the genomic level about the identity of the disease-causing pathogens and their pathogenesis, which help researchers, healthcare professionals, and patients to detect the presence of pathogens, track the spread of disease, and guide treatment more efficiently. A typical nucleic acid-based diagnostic test consists of three major steps: nucleic acid extraction, amplification, and amplicon detection. Among these steps, nucleic acid extraction is the first step of sample preparation, which remains one of the main challenges when converting laboratory molecular assays into POC tests. Sample preparation from human and plant specimens is a time-consuming and multi-step process, which requires well-equipped laboratories and skilled lab personnel. To perform rapid molecular diagnosis in resource-limited settings, simpler and instrument-free nucleic acid extraction techniques are required to improve the speed of field detection with minimal human intervention. This review summarizes the recent advances in POC nucleic acid extraction technologies. In particular, this review focuses on novel devices or methods that have demonstrated applicability and robustness for the isolation of high-quality nucleic acid from complex raw samples, such as human blood, saliva, sputum, nasal swabs, urine, and plant tissues. The integration of these rapid nucleic acid preparation methods with miniaturized assay and sensor technologies would pave the road for the "sample-in-result-out" diagnosis of human and plant diseases, especially in remote or resource-limited settings.


Assuntos
Doenças Transmissíveis/diagnóstico , Dispositivos Lab-On-A-Chip , Ácidos Nucleicos/isolamento & purificação , Doenças das Plantas , Sistemas Automatizados de Assistência Junto ao Leito , Betacoronavirus/isolamento & purificação , Fracionamento Químico/instrumentação , Fracionamento Químico/métodos , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/parasitologia , Doenças Transmissíveis/virologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Desenho de Equipamento , Humanos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Pandemias , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Doenças das Plantas/virologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia
4.
Public Health ; 186: 1-5, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32731151

RESUMO

OBJECTIVES: Nucleic acid testing is the gold standard method for the diagnosis of coronavirus disease 2019 (COVID-19); however, large numbers of false-negative results have been reported. In this study, nucleic acid detection and antibody detection (IgG and IgM) were combined to improve the testing accuracy of patients with suspected COVID-19. STUDY DESIGN: The positive rate of nucleic acid detection and antibody detection (IgG and IgM) were compared in suspected COVID-19 patients. METHODS: A total of 71 patients with suspected COVID-19 were selected to participate in this study, which included a retrospective analysis of clinical features, imaging examination, laboratory biochemical examination and nucleic acid detection and specific antibody (IgM and IgG) detection. RESULTS: The majority of participants with suspected COVID-19 presented with fever (67.61%) and cough (54.93%), and the imaging results showed multiple small patches and ground-glass opacity in both lungs, with less common infiltration and consolidation opacity (23.94%). Routine blood tests were mostly normal (69.01%), although only a few patients had lymphopenia (4.23%) or leucopenia (12.68%). There was no statistical difference in the double-positive rate between nucleic acid detection (46.48%) and specific antibody (IgG and IgM) detection (42.25%) (P = 0.612), both of which were also poorly consistent with each other (kappa = 0.231). The positive rate of combined nucleic acid detection and antibody detection (63.38%) was significantly increased, compared with that of nucleic acid detection (46.48%) and that of specific antibody (IgG and IgM) detection (42.25%), and the differences were statistically significant (P = 0.043 and P = 0.012, respectively). CONCLUSIONS: Nucleic acid detection and specific antibody (IgG and IgM) detection had similar positive rates, and their combination could improve the positive rate of COVID-19 detection, which is of great significance for diagnosis and epidemic control.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Adolescente , Adulto , Idoso , Anticorpos Antivirais/isolamento & purificação , Betacoronavirus/genética , Betacoronavirus/imunologia , Criança , Pré-Escolar , Infecções por Coronavirus/epidemiologia , Feminino , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina M/isolamento & purificação , Masculino , Pessoa de Meia-Idade , Ácidos Nucleicos/isolamento & purificação , Pandemias , Pneumonia Viral/epidemiologia , Reprodutibilidade dos Testes , Estudos Retrospectivos , Adulto Jovem
5.
J Nanobiotechnology ; 18(1): 62, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32316985

RESUMO

Nucleic acid is the main material for storing, copying, and transmitting genetic information. Gene sequencing is of great significance in DNA damage research, gene therapy, mutation analysis, bacterial infection, drug development, and clinical diagnosis. Gene detection has a wide range of applications, such as environmental, biomedical, pharmaceutical, agriculture and forensic medicine to name a few. Compared with Sanger sequencing, high-throughput sequencing technology has the advantages of larger output, high resolution, and low cost which greatly promotes the application of sequencing technology in life science research. Magnetic nanoparticles, as an important part of nanomaterials, have been widely used in various applications because of their good dispersion, high surface area, low cost, easy separation in buffer systems and signal detection. Based on the above, the application of magnetic nanoparticles in nucleic acid detection was reviewed.


Assuntos
Nanopartículas de Magnetita/química , Ácidos Nucleicos/análise , Bactérias/genética , DNA Bacteriano/análise , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Óxido Ferroso-Férrico/química , Humanos , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/isolamento & purificação , Polimorfismo de Nucleotídeo Único
6.
Sci Rep ; 10(1): 1940, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029846

RESUMO

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2-2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Transcrição Reversa/fisiologia , Sensibilidade e Especificidade
7.
Biosens Bioelectron ; 151: 112002, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999596

RESUMO

Herein, an isothermal padlock probe-based assay for the simple and portable detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical readout is reported. Infectious diseases remain a constant threat on a global scale, as in recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralising healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. The design of the demonstrated assay consists of a rolling circle amplification (RCA) technique, responsible for the generation of nucleic acid amplicons as RCA products (RCPs). The RCPs are generated on magnetic beads (MB) and subsequently, connected via streptavidin-biotin bonds to GOx. The enzymatic catalysis of glucose by the bound GOx allows for an indirect electrochemical measurement of the DNA target. The RCPs generated on the surface of the MB were confirmed by scanning electron microscopy, and among other experimental conditions such as the type of buffer, temperature, concentration of GOx, sampling and measurement time were evaluated for the optimum electrochemical detection. Accordingly, 125 µg mL-1 of GOx with 5 mM glucose using phosphate buffer saline (PBS), monitored for 1 min were selected as the ideal conditions. Finally, we assessed the analytical performance of the biosensing strategy by using clinical samples of Ebola virus from patients. Overall, this work provides a proof-of-concept bioassay for simple and portable molecular diagnostics of emerging pathogens using electrochemical detection, especially in resource-limited settings.


Assuntos
Técnicas Biossensoriais , DNA Viral/isolamento & purificação , Técnicas Eletroquímicas , Ácidos Nucleicos/isolamento & purificação , DNA Viral/genética , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Ebolavirus/patogenicidade , Glucose/química , Glucose Oxidase/química , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/genética
8.
Biosens Bioelectron ; 147: 111762, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31654822

RESUMO

We, herein, describe a three-way junction (3WJ)-induced isothermal amplification (ThIsAmp) reaction for target nucleic acid detection. In this strategy, target nucleic acid induces the formation of 3WJ structure by associating a specially designed ThIsAmp template and ThIsAmp primer. Upon the formation of 3WJ structure, ThIsAmp primer is subjected to continuously repeated extension and nicking reaction by the combined activities of DNA polymerase and nicking endonuclease, consequently producing a large number of trigger strands. The trigger strands then initiate two separate but interconnected pathways by binding to either 3' overhang of ThIsAmp template within the 3WJ structure or free ThIsAmp template. As a consequence, a large number of final double-stranded DNA products are produced under an isothermal condition, which can be monitored in real-time by detecting the fluorescence intensity resulting from SYBR Green I staining. Based on this principle, we successfully detected target DNA down to 78.1 aM with excellent specificity. The sophisticated design principle employed in this work would provide great insight for the development of self-operative isothermal amplifying system enabling target nucleic acid detection.


Assuntos
Técnicas Biossensoriais , DNA Polimerase Dirigida por DNA/química , DNA/química , Ácidos Nucleicos/isolamento & purificação , DNA/genética , Quebras de DNA de Cadeia Simples , Fluorescência , Ácidos Nucleicos/química , Compostos Orgânicos/química , Espectrometria de Fluorescência
9.
Int J Legal Med ; 134(1): 149-157, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31773316

RESUMO

Postmortem interval (PMI) determination is an important part of criminal investigations, but it is still subject to uncertainty. Degradation of mRNA in PMI determination has been studied in decays; however, some studies have reported no correlation between PMI and RNA degradation. Thus, we aimed to determine whether RNA quantity was correlated with PMI. Heart and brain tissues were separated from a mouse model of a 0-48 h PMI with 29 time points. We then coextracted the DNA and RNA in one tube with Bioteke coextraction kits and selected some mRNA markers associated with cell oxygen deprivation and apoptosis as target genes, such as hypoxia-associated factor (HAF), apoptosis-inducing factor (AIF), hypoxia-inducible factor 2 alpha (HIF2a), and factor inhibiting HIF (FIH). We measured the quantity of these markers using real-time quantitative PCR (qPCR), and Caspase-3 DNA and 18S were each used for normalization. The results showed that in the heart tissue, the degradation of HIF2a, AIF, and FIH was correlated with PMI, as was the degradation of HIF2a, FIH, and AIF in brain tissue when normalized with Caspase-3 DNA. However, when normalized with 18S, only the degradation of HIF2a in brain tissue was correlated with PMI. Interestingly, the quantity of HAF in brain tissue was found to increase after death with either 18S or Caspase-3 DNA normalization, and it was significantly correlated with 0-48 h PMI. These results indicated that mRNA quantity can be used to determine PMI and that Caspase-3 DNA is feasible for PMI estimation. In summary, we established mathematical models for PMI determination using multiple mRNA markers and multiple tissues and further studies are needed to validate and investigate these markers and mathematical models in human tissues.Duo Peng and Meili Lv contributed equally to this work.


Assuntos
Biomarcadores/análise , Mudanças Depois da Morte , Estabilidade de RNA , RNA Mensageiro/análise , Animais , Fator de Indução de Apoptose/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Encéfalo/metabolismo , Caspase 3/genética , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Oxigenases de Função Mista/genética , Modelos Animais , Modelos Teóricos , Miocárdio/metabolismo , Ácidos Nucleicos/isolamento & purificação , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Ribonucleoproteínas Nucleares Pequenas/genética
10.
Sensors (Basel) ; 19(23)2019 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-31795306

RESUMO

Information about the characteristics of measuring chips according to their storage conditions is of great importance for clinical diagnosis. In our present work, we have studied the capability of chips to detect nanowire biosensors when they are either freshly prepared or have been stored for either one or two years in a clean room. Potential to detect DNA oligonucleotides (oDNAs)-synthetic analogues of microRNAs (miRNAs) 198 and 429 that are associated with the development of prostate cancer (PCa)-in buffer solution was demonstrated using a nanowire biosensor based on silicon-on-insulator structures (SOI-NW biosensor). To provide biospecific detection, nanowire surfaces were sensitized with oligonucleotide probes (oDNA probes) complimentary to the known sequences of miRNA 183 and 484. In this study it is demonstrated that freshly prepared SOI-NW biosensor chips with n-type conductance and immobilized oDNA probes exhibit responses to the addition of complimentary oDNAs in buffer, leading to decreases in chips' conductance at a concentration of 3.3 × 10-16 M. The influence of storage time on the characteristics of SOI-NW biosensor chips is also studied herein. It is shown that a two-year storage of the chips leads to significant changes in their characteristics, resulting in "inverse" sensitivity toward negatively charged oDNA probes (i.e., through an increase in chips' conductance). It is concluded that the surface layer makes the main contribution to conductance of the biosensor chip. Our results indicate that the detection of target nucleic acid molecules can be carried out with high sensitivity using sensor chips after long-term storage, but that changes in their surface properties, which lead to inversed detection signals, must be taken into account. Examples of the applications of such chips for the detection of cancer-associated microRNAs in plasma samples of patients with diagnosed prostate cancer are given. The results obtained herein are useful for the development of highly sensitive nanowire-based diagnostic systems for the revelation of (prostate) cancer-associated microRNAs in human plasma.


Assuntos
Biomarcadores Tumorais/sangue , Técnicas Biossensoriais/métodos , MicroRNAs/sangue , Neoplasias da Próstata/sangue , Humanos , Masculino , Nanofios/química , Ácidos Nucleicos/sangue , Ácidos Nucleicos/isolamento & purificação , Silício/química
11.
J Biosci ; 44(5)2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31719232

RESUMO

The importance of studying microbial load on fabrics has been recently realized with reports on fabrics being a source of spread of infection in medical and hospitality sectors. However, methodological limitations have restricted the analysis of microbial diversity on fabrics. Hence, the study aimed to develop a robust method for extraction of DNA from different types of fabrics. Bacterial community profiles could be successfully generated with DNA extracted from real life samples, together with identification of different bacterial genera on fabrics. The study opens up venues to study effect of environmental factors on microbial load on fabrics. Also, such a technique will aid correlation between microbial load and types of fabric so as to come up with recommendation for fabrics bearing minimal microbial load for medical and hospitality sectors.


Assuntos
Bactérias/classificação , Ácidos Nucleicos/isolamento & purificação , Têxteis , Filogenia , Especificidade da Espécie
12.
Sci Rep ; 9(1): 16374, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31705044

RESUMO

Here, we describe a simple, universal protocol for use in nucleic acid testing-based pathogen diagnostics, which requires only hand-powered sample preparation, including the processes of pathogen enrichment and nucleic acid isolation. The protocol uses low-cost amine-functionalized diatomaceous earth with a 1-µm Teflon filter as a reaction matrix in both stages of the process, using homobifunctional imidoesters. Using a simple syringe as a pump, the capture efficiency for a large sample volume (<50 mL) was enhanced by up to 98.3%, and the detection limit was 1 CFU/mL, 100-fold better than that of common commercial nucleic acid isolation kit. This protocol can also be combined with commercialized 96-well filter plates for robust sample preparation. Our proposed system is robust, simple, low-cost, universal, and rapid (taking <20 min), and it works regardless of the ambient environment and sample pretreatment, requiring no electricity or instruments. Its benefits include the simplicity of producing its components and its ease of operation, and it can be readily integrated with other assays for point-of-care diagnostics.


Assuntos
Ácidos Nucleicos/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Aspergillus fumigatus/genética , Aspergillus fumigatus/isolamento & purificação , Brucella ovis/genética , Brucella ovis/isolamento & purificação , Equipamentos para Diagnóstico , Terra de Diatomáceas , Desenho de Equipamento , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/instrumentação , Ácidos Nucleicos/genética , Reação em Cadeia da Polimerase em Tempo Real , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação
13.
Lab Chip ; 19(22): 3853-3861, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31621762

RESUMO

We report a bifurcated continuous field-flow fractionation (BCFFF) chip for high-yield and high-throughput (20 min) extraction of nucleic acids from physiological samples. The design uses a membrane ionic transistor to sustain low-ionic strength in a localized region at a junction, such that the resulting high field can selectively isolate high-charge density nucleic acids from the main flow channel and insert them into a standardized buffer in a side channel that bifurcates from the junction. The high local electric field and the bifurcated field-flow design facilitate concentration reduction of both divalent cation (Ca2+) and molecular PCR inhibitors by more than two orders of magnitude, even with high-throughput continuous loading. The unique design with a large (>20 mM mm-1) on-chip ionic-strength gradient allows miniaturization into a high-throughput field-flow fractionation chip that can be integrated with upstream lysing and downstream PCR/sensor modules for various nucleic acid detection/quantification applications. A concentration-independent 85% yield for extraction and an overall post-PCR yield exceeding 60% are demonstrated for a 111 bp dsDNA in 10 µL of human plasma, compared to no amplification with the raw sample. A net yield four times larger than a commercial extraction kit is demonstrated for miR-39 in human plasma.


Assuntos
Fracionamento por Campo e Fluxo , Ensaios de Triagem em Larga Escala , Técnicas Analíticas Microfluídicas , Ácidos Nucleicos/isolamento & purificação , Cálcio/sangue , Fracionamento por Campo e Fluxo/instrumentação , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Ácidos Nucleicos/sangue , Ácidos Nucleicos/genética , Reação em Cadeia da Polimerase
14.
PLoS Biol ; 17(8): e3000374, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393866

RESUMO

A deep understanding of how regulation of the multiple levels of gene expression in mammalian tissues give rise to complex phenotypes has been impeded by cellular diversity. A handful of techniques were developed to tag-select nucleic acids of interest in specific cell types, thereby enabling their capture. We expanded this strategy by developing the Tagger knock-in mouse line bearing a quad-cistronic transgene combining enrichment tools for nuclei, nascent RNA, translating mRNA, and mature microRNA (miRNA). We demonstrate that Tagger can capture the desired nucleic acids, enabling multiple omics approaches to be applied to specific cell types in vivo using a single transgenic mouse line.


Assuntos
Perfilação da Expressão Gênica/métodos , Ácidos Nucleicos/isolamento & purificação , Sequenciamento Completo do Genoma/métodos , Animais , Clonagem Molecular/métodos , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Técnicas de Introdução de Genes , Genômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , MicroRNAs/genética , Proteômica/métodos , RNA Mensageiro/genética , Transcriptoma/genética , Transgenes/genética
15.
Lab Chip ; 19(17): 2769-2785, 2019 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-31365009

RESUMO

Rapid, efficient and accurate nucleic acid molecule detection is important in the screening of diseases and pathogens, yet remains a limiting factor at point of care (POC) treatment. Microfluidic systems are characterized by fast, integrated, miniaturized features which provide an effective platform for qualitative and quantitative detection of nucleic acid molecules. The nucleic acid detection process mainly includes sample preparation and target molecule amplification. Given the advancements in theoretical research and technological innovations to date, nucleic acid extraction and amplification integrated with microfluidic systems has advanced rapidly. The primary goal of this review is to outline current approaches used for nucleic acid detection in the context of microfluidic systems. The secondary goal is to identify new approaches that will help shape future trends at the intersection of nucleic acid detection and microfluidics, particularly with regard to increasing disease and pathogen detection for improved diagnosis and treatment.


Assuntos
Técnicas Analíticas Microfluídicas , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/isolamento & purificação , Sistemas Automatizados de Assistência Junto ao Leito , Humanos , Ácidos Nucleicos/química
16.
Lab Chip ; 19(18): 2973-2977, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31433426

RESUMO

Here, we report an integrated operation of microfluidic pumps and valves only by finger actuation. As the working principle of the finger-actuated microfluidic pumps includes deflection of the poly(dimethylsiloxane) (PDMS) membrane, the pneumatic valves for controlling the flow direction can be easily integrated with the pumps. Using a single button, the flow path can be determined and flow generation can be achieved. We also verified the integrated operation of finger-actuated pumps and valves by demonstrating nucleic acid purification.


Assuntos
Técnicas Analíticas Microfluídicas , DNA Viral/química , DNA Viral/isolamento & purificação , Dimetilpolisiloxanos/química , Vírus da Hepatite B/química , Técnicas Analíticas Microfluídicas/instrumentação , Ácidos Nucleicos/química , Ácidos Nucleicos/isolamento & purificação
17.
J Virol Methods ; 273: 113715, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31419455

RESUMO

Detection and quantification of viral nucleic acids are important for diagnosing current viral infections and monitoring response to antiviral therapy. Automated nucleic acid extraction and purification platforms are routinely used during the first step in these processes in clinical and research laboratories. Here, we compare the extraction efficiencies of four MagNA Pure magnetic bead-based nucleic acid extraction platforms and associated kits using samples positive for nucleic acids from HAV, HBV, HCV, HDV, and HEV. These five hepatitis viruses are diverse in their virion structures and type of nucleic acid that compose their genomes. We found that the most efficient nucleic acid extraction platform and corresponding kit, when averaged across all tested viruses, was the MagNA Pure 96, which yielded twice as much detectable nucleic acid as the other platforms. However, the relative efficiencies of the different platforms varied by virus type, suggesting that an extraction platform that is more efficient for one virus type will not necessarily function better with a different virus type. Our results show that the choice of a nucleic acid extraction platform influences the sensitivity of the methodology and has the potential to generate false-negative results especially in samples with low levels of viral nucleic acids.


Assuntos
Genoma Viral , Vírus de Hepatite/isolamento & purificação , Ácidos Nucleicos/isolamento & purificação , Kit de Reagentes para Diagnóstico/normas , DNA Viral/isolamento & purificação , Hepatite Viral Humana , Humanos , Ácidos Nucleicos/sangue , RNA Viral/isolamento & purificação , Sensibilidade e Especificidade
18.
Proc Natl Acad Sci U S A ; 116(33): 16240-16249, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31358642

RESUMO

Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g., PCR), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis. We report a universal biomolecule enrichment technique termed hierarchical nanofluidic molecular enrichment system (HOLMES) for amplification-free molecular diagnostics using massively paralleled and hierarchically cascaded nanofluidic concentrators. HOLMES achieves billion-fold enrichment of both nucleic acids and proteins within 30 min, which not only overcomes many inherent issues of nucleic acid amplification but also provides unprecedented enrichment performance for protein analysis. HOLMES features the ability to selectively enrich target biomolecules and simultaneously deplete nontargets directly in complex crude samples, thereby enormously enhancing the signal-to-noise ratio of detection. We demonstrate the direct detection of attomolar nucleic acids in urine and serum within 35 min and HIV p24 protein in serum within 60 min. The performance of HOLMES is comparable to that of nucleic acid amplification tests and near million-fold improvement over standard enzyme-linked immunosorbent assay (ELISA) for protein detection, being much simpler and faster in both applications. We additionally measured human cardiac troponin I protein in 9 human plasma samples, and showed excellent agreement with ELISA and detection below the limit of ELISA. HOLMES is in an unparalleled position to unleash the potential of protein-based diagnosis.


Assuntos
Proteínas Sanguíneas/isolamento & purificação , Nanotecnologia/tendências , Ácidos Nucleicos/isolamento & purificação , Patologia Molecular/métodos , Proteínas Sanguíneas/química , Ensaio de Imunoadsorção Enzimática , Proteína do Núcleo p24 do HIV/sangue , Proteína do Núcleo p24 do HIV/isolamento & purificação , Proteína do Núcleo p24 do HIV/urina , Humanos , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Troponina I/sangue , Troponina I/isolamento & purificação
19.
Artigo em Inglês | MEDLINE | ID: mdl-31176269

RESUMO

Plant polyphenols can form functional coatings on various materials through self-polymerization. In this paper, a series of modified capillary columns, which possess diversity of charge characteristics for modulating electroosmotic flow (EOF), were prepared by one-step co-deposition of gallic acid (GA), a plant-derived polyphenol monomer, and branched polyethyleneimine (PEI). The physicochemical properties of the prepared columns were characterized by Fourier transform infrared spectroscopy (FT-IR), UV-Vis spectroscopy and scanning electron microscopy (SEM). The magnitude and direction of EOF of GA/PEI co-deposited columns were modulated by changing a series of coating parameters, such as post-incubation of FeCl3, co-deposition time, and deposited amounts of GA and PEI with different relative molecular mass (PEI-600, PEI-1800, PEI-10000, and PEI-70000). Furthermore, the separation efficiencies of the prepared GA/PEI co-deposited columns were evaluated by separations of small molecules, including organic acids, polar nucleotides, phenols, nucleic acid bases and nucleosides. Results indicated that modulating of EOF plays an important role in enhancing the separation performance and reversing the elution order of the analytes. Finally, the developed method was successfully applied to quantitative analysis of acidic compounds in four real samples. The recoveries were in the range of 73.5%-85.8% for citric acid, benzoic acid, sorbic acid, salicylic acid and ascorbic acid in beverage and fruit samples, 101.6%-104.9% for cinnamic acid, vanillic acid, and ferulic acid in Angelica sinensis sample, while 84.6%-97.8% for guanosine-5'-monophosphate, uridine-5'-monophosphate, cytosine-5'- monophosphate and adenosine-5'-monophosphate in Cordyceps samples. These results indicated that the co-deposition of plant polyphenol-inspired GA/PEI coatings can provide new opportunities for EOF modulation of capillary electrophoresis.


Assuntos
Eletrocromatografia Capilar/métodos , Eletro-Osmose/métodos , Ácido Gálico/química , Polietilenoimina/química , Eletrocromatografia Capilar/instrumentação , Eletro-Osmose/instrumentação , Peso Molecular , Ácidos Nucleicos/isolamento & purificação , Nucleosídeos/isolamento & purificação , Nucleotídeos/isolamento & purificação , Compostos Orgânicos/isolamento & purificação , Polimerização
20.
Astrobiology ; 19(9): 1139-1152, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31204862

RESUMO

Recent studies regarding the origins of life and Mars-Earth meteorite transfer simulations suggest that biological informational polymers, such as nucleic acids (DNA and RNA), have the potential to provide unambiguous evidence of life on Mars. To this end, we are developing a metagenomics-based life-detection instrument which integrates nucleic acid extraction and nanopore sequencing: the Search for Extra-Terrestrial Genomes (SETG). Our goal is to isolate and sequence nucleic acids from extant or preserved life on Mars in order to determine if a particular genetic sequence (1) is distantly related to life on Earth, indicating a shared ancestry due to lithological exchange, or (2) is unrelated to life on Earth, suggesting convergent origins of life on Mars. In this study, we validate prior work on nucleic acid extraction from cells deposited in Mars analog soils down to microbial concentrations (i.e., 104 cells in 50 mg of soil) observed in the driest and coldest regions on Earth. In addition, we report low-input nanopore sequencing results from 2 pg of purified Bacillus subtilis spore DNA simulating ideal extraction yields equivalent to 1 ppb life-detection sensitivity. We achieve this by employing carrier sequencing, a method of sequencing sub-nanogram DNA in the background of a genomic carrier. After filtering of carrier, low-quality, and low-complexity reads we detected 5 B. subtilis reads, 18 contamination reads (including Homo sapiens), and 6 high-quality noise reads believed to be sequencing artifacts.


Assuntos
Biomassa , Exobiologia/métodos , Marte , Ácidos Nucleicos/isolamento & purificação , Análise de Sequência de DNA , Solo/química , DNA/análise , DNA/isolamento & purificação , Humanos , Sequenciamento por Nanoporos , Esporos Bacterianos/genética , Água/química
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