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Circular RNAs (circRNAs) have emerged as pivotal players in RNA therapeutics. Unlike linear counterparts, circRNAs possess a closed-loop structure, conferring them with enhanced stability and resistance to degradation. Ribozyme-based strategy stands out as the predominant method for synthetic circRNA production, by precisely cleaving and promoting the formation of a covalent circular structure. However, there is still a lack of analytical methods that can provide high-throughput and quantitative analysis to facilitate the circRNA vector engineering process. In the report, we detail analytical methods to characterize and evaluate ribozyme-based RNA circularization efficiency. Our approach will capture the attention of researchers interested in optimizing RNA circularization efficiency, as well as those focused on exploring key elements for ribozyme catalytic activity.
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Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at â¼89 pg/µL (3 × 1010 GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at â¼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.
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ADN de Cadena Simple , Dependovirus , Electroforesis Capilar , Genoma Viral , Dependovirus/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/análisis , Genoma Viral/genética , Electroforesis Capilar/métodos , Tamaño del Genoma , Humanos , ADN Viral/genética , ADN Viral/análisis , Estándares de Referencia , Técnicas Analíticas Microfluídicas/métodosRESUMEN
Quantitative diagnostics that are rapid, inexpensive, sensitive, robust, and field-deployable are needed to contain the spread of infectious diseases and inform treatment strategies. While current gold-standard techniques are highly sensitive and quantitative, they are slow and require expensive equipment. Conversely, current rapid field-deployable assays available provide essentially binary information about the presence of the target analyte, not a quantitative measure of concentration. Here, we report the development of a molecular diagnostic test [quantitative recombinase polymerase amplification (qRPA)] that utilizes competitive amplification during a recombinase polymerase amplification (RPA) assay to provide semi-quantitative information on a target nucleic acid. We demonstrate that qRPA can quantify DNA, RNA, and viral titers in HIV and COVID-19 patient samples and that it is more robust to environmental perturbations than traditional RPA. These features make qRPA potentially useful for at-home testing to monitor the progress of viral infections or other diseases.
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COVID-19 , Técnicas de Amplificación de Ácido Nucleico , Humanos , Técnicas de Diagnóstico Molecular , Recombinasas , SARS-CoV-2 , Sensibilidad y EspecificidadRESUMEN
Photochromic materials are constructed with molecules accompanied by structural change after triggering by light, which are of great importance and necessity for various applications. However, because of space-confinement effects, molecule stacking of these photoresponsive chromophores within coordination polymers (CPs) always results in an efficiency decrement and a response delay, and this phenomenon will lead to a poor photochromic property. Herein, a CP (named CIT-E) with a 3-fold-interpenetrating network structure, which was prepared with (Z)-1,2-diphenyl-1,2-bis[4-(pyridin-3-ylmethoxy)phenyl]ethene (1Z) and a CuI cluster, showed fast reversible photochromic behavior. Under UV-light illumination, the color of CIT-Z changed from pale yellow to reddish brown. With the illumination of green light, the polymer could return to its initial color within 10 s. To reveal the mechanism of reversible photochromic behavior of CIT-Z, single-crystal structures of each color state were fully studied, and other scientific study methods were also used, such as time-dependent density functional theory calculation and control experiments. It was found that, with light illumination, this behavior of CIT-Z was the result of a ligand-to-metal charge-transfer process, and this process was triggered by subtle molecular conformation variation of tetraphenylethylene. It should be noted that CIT-Z has high thermal and chemical stability, which are excellent advantages as smart photoresponsive materials. As a proof of concept, a uniform thin film with such a fascinating photochromic property allows applications in invisible anticounterfeiting and dynamic optical data storage. Overall, the present study opens up a new avenue toward reversible photochromic materials.
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N(6)-Methyladenosine (m(6)A) is a widespread, reversible chemical modification of RNA molecules, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m(6)A modified ('m(6)A levels') or the relationship of m(6)A modification(s) to alternative RNA isoforms. To deconvolute the m(6)A epitranscriptome, we developed m(6)A-level and isoform-characterization sequencing (m(6)A-LAIC-seq). We found that cells exhibit a broad range of nonstoichiometric m(6)A levels with cell-type specificity. At the level of isoform characterization, we discovered widespread differences in the use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3' untranslated regions, while nonmethylated transcript isoforms tend to use distal APA sites. m(6)A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m(6)A modifications.
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Adenosina/análogos & derivados , Epigénesis Genética/genética , Epigenómica/métodos , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Transcriptoma , Regiones no Traducidas 3'/genética , Adenosina/química , Adenosina/genética , Células Cultivadas , Humanos , Poliadenilación , Isoformas de Proteínas , ARN Mensajero/genética , Células Madre/metabolismoRESUMEN
Over the last decade, the controllable reversible phase transition of functional materials has received growing interest as it shows unique suitability for various technological applications. Although many metal-organic frameworks (MOFs) possess a lamellar structure, the reversible structural transformation of MOFs between their three-dimensional (3D) phase and two-dimensional (2D) phase remains a largely unexplored area. Herein, we report for the first time a europium MOF with unprecedented reversible morphology in different solvents at room temperature. This europium MOF displayed a 3D nanorod morphology in organic solvent and a 2D nanobelt architecture in water. As a proof of concept for potential applications of this reversible-phase-transition MOF, we were able to use a delamination recovery method to load dye molecules that previously could not be loaded into europium MOFs.
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A series of silver coordination polymers (CPs) have been synthesized through self-assembly of three pyridinecarboxylic acid hydrazide (p-, m-, o-position) ligands with silver clusters (named Ag1-iah, Ag2-iah, and Ag3-iah). These silver CPs show different one- and two-dimensional topologies including cross-helical chains, planar network, and parallel helical chains for Ag1-iah, Ag2-iah, and Ag3-iah, respectively. The combination of experimental and computational results reveals the critical role in the space distribution of the coordination site of silver clusters and ligands in controlling the silver CPs' dimensionality and packing arrangement and modulating the optical properties and stability. Luminescent investigations reveal that Ag3-iah can selectively detect dichloromethane or trichloromethane in tetrachloromethane. These silver CPs provide a good model to study the influence of the space distribution of the coordination site of ligands on their packing arrangement and properties.
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Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)2D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)2D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)2D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)2D-mediated induction of transcription.
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Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Osteocitos/metabolismo , Empalme del ARN , Transcripción Genética , Vitamina D/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Análisis por Conglomerados , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Osteocitos/efectos de los fármacos , Regiones Promotoras Genéticas , Unión Proteica , Interferencia de ARN , Precursores del ARN , Vitamina D3 24-Hidroxilasa/genética , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
RNA-seq has become a popular technology for studying genetic variation of pre-mRNA alternative splicing. Commonly used RNA-seq aligners rely on the consensus splice site dinucleotide motifs to map reads across splice junctions. Consequently, genomic variants that create novel splice site dinucleotides may produce splice junction RNA-seq reads that cannot be mapped to the reference genome. We developed and evaluated an approach to identify 'hidden' splicing variations in personal transcriptomes, by mapping personal RNA-seq data to personal genomes. Computational analysis and experimental validation indicate that this approach identifies personal specific splice junctions at a low false positive rate. Applying this approach to an RNA-seq data set of 75 individuals, we identified 506 personal specific splice junctions, among which 437 were novel splice junctions not documented in current human transcript annotations. 94 splice junctions had splice site SNPs associated with GWAS signals of human traits and diseases. These involve genes whose splicing variations have been implicated in diseases (such as OAS1), as well as novel associations between alternative splicing and diseases (such as ICA1). Collectively, our work demonstrates that the personal genome approach to RNA-seq read alignment enables the discovery of a large but previously unknown catalog of splicing variations in human populations.
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Empalme Alternativo , Perfilación de la Expresión Génica/métodos , Genoma Humano , Polimorfismo de Nucleótido Simple , Sitios de Empalme de ARN , Análisis de Secuencia de ARN/métodos , Enfermedad/genética , Estudio de Asociación del Genoma Completo , Humanos , TranscriptomaRESUMEN
Ultra-deep RNA sequencing (RNA-Seq) has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We previously developed multivariate analysis of transcript splicing (MATS), a statistical method for detecting differential alternative splicing between two RNA-Seq samples. Here we describe a new statistical model and computer program, replicate MATS (rMATS), designed for detection of differential alternative splicing from replicate RNA-Seq data. rMATS uses a hierarchical model to simultaneously account for sampling uncertainty in individual replicates and variability among replicates. In addition to the analysis of unpaired replicates, rMATS also includes a model specifically designed for paired replicates between sample groups. The hypothesis-testing framework of rMATS is flexible and can assess the statistical significance over any user-defined magnitude of splicing change. The performance of rMATS is evaluated by the analysis of simulated and real RNA-Seq data. rMATS outperformed two existing methods for replicate RNA-Seq data in all simulation settings, and RT-PCR yielded a high validation rate (94%) in an RNA-Seq dataset of prostate cancer cell lines. Our data also provide guiding principles for designing RNA-Seq studies of alternative splicing. We demonstrate that it is essential to incorporate biological replicates in the study design. Of note, pooling RNAs or merging RNA-Seq data from multiple replicates is not an effective approach to account for variability, and the result is particularly sensitive to outliers. The rMATS source code is freely available at rnaseq-mats.sourceforge.net/. As the popularity of RNA-Seq continues to grow, we expect rMATS will be useful for studies of alternative splicing in diverse RNA-Seq projects.
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Empalme Alternativo , Simulación por Computador , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Línea Celular Tumoral , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
Changes in exon-intron structures and splicing patterns represent an important mechanism for the evolution of gene functions and species-specific regulatory networks. Although exon creation is widespread during primate and human evolution and has been studied extensively, much less is known about the scope and potential impact of human-specific exon loss events. Historically, transcriptome data and exon annotations are significantly biased toward humans over nonhuman primates. This ascertainment bias makes it challenging to discover human-specific exon loss events. We carried out a transcriptome-wide search of human-specific exon loss events, by taking advantage of RNA sequencing (RNA-seq) as a powerful and unbiased tool for exon discovery and annotation. Using RNA-seq data of humans, chimpanzees, and other primates, we reconstructed and compared transcript structures across the primate phylogeny. We discovered 33 candidate human-specific exon loss events, among which six exons passed stringent experimental filters for the complete loss of splicing activities in diverse human tissues. These events may result from human-specific deletion of genomic DNA, or small-scale sequence changes that inactivated splicing signals. The impact of human-specific exon loss events is predominantly regulatory. Three of the six events occurred in the 5' untranslated region (5'-UTR) and affected cis-regulatory elements of mRNA translation. In SLC7A6, a gene encoding an amino acid transporter, luciferase reporter assays suggested that both a human-specific exon loss event and an independent human-specific single nucleotide substitution in the 5'-UTR increased mRNA translational efficiency. Our study provides novel insights into the molecular mechanisms and evolutionary consequences of exon loss during human evolution.
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Exones/genética , Transcriptoma/genética , Sistemas de Transporte de Aminoácidos Básicos/genética , Animales , Evolución Molecular , Humanos , Primates/genéticaRESUMEN
Ultra-deep RNA sequencing has become a powerful approach for genome-wide analysis of pre-mRNA alternative splicing. We develop MATS (multivariate analysis of transcript splicing), a bayesian statistical framework for flexible hypothesis testing of differential alternative splicing patterns on RNA-Seq data. MATS uses a multivariate uniform prior to model the between-sample correlation in exon splicing patterns, and a Markov chain Monte Carlo (MCMC) method coupled with a simulation-based adaptive sampling procedure to calculate the P-value and false discovery rate (FDR) of differential alternative splicing. Importantly, the MATS approach is applicable to almost any type of null hypotheses of interest, providing the flexibility to identify differential alternative splicing events that match a given user-defined pattern. We evaluated the performance of MATS using simulated and real RNA-Seq data sets. In the RNA-Seq analysis of alternative splicing events regulated by the epithelial-specific splicing factor ESRP1, we obtained a high RT-PCR validation rate of 86% for differential exon skipping events with a MATS FDR of <10%. Additionally, over the full list of RT-PCR tested exons, the MATS FDR estimates matched well with the experimental validation rate. Our results demonstrate that MATS is an effective and flexible approach for detecting differential alternative splicing from RNA-Seq data.
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Empalme Alternativo , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ARN , Teorema de Bayes , Encéfalo/metabolismo , Línea Celular Tumoral , Humanos , Análisis Multivariante , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
There has been growing evidence for extensive diversity of alternative splicing in human populations. Genetic variants within the 5' splice site can cause splicing differences among human individuals and constitute an important class of human disease mutations. In this study, we explored whether natural variations of splicing could reveal important signals of 5' splice site recognition. In seven lymphoblastoid cell lines of Asian, European and African ancestry, we identified 1174 single nucleotide polymorphisms (SNPs) within the consensus 5' splice site. We selected 129 SNPs predicted to significantly alter the splice site activity, and quantitatively examined their splicing impact in the seven individuals. Surprisingly, outside of the essential GT dinucleotide position, only â¼14% of the tested SNPs altered splicing. Bioinformatic and minigene analyses identified signals that could modify the impact of 5' splice site polymorphisms, most notably a strong 3' splice site and the presence of intronic motifs downstream of the 5' splice site. Strikingly, we found that the poly-G run, a known intronic splicing enhancer, was the most significantly enriched motif downstream of exons unaffected by 5' splice site SNPs. In TRIM62, the upstream 3' splice site and downstream intronic poly-G runs functioned redundantly to protect an exon from its 5' splice site polymorphism. Collectively, our study reveals widespread context-dependent robustness to 5' splice site polymorphisms in human transcriptomes. Consequently, certain exons are more susceptible to 5' splice site mutations. Additionally, our work demonstrates that genetic diversity of alternative splicing can provide significant insights into the splicing code of mammalian cells.
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Polimorfismo de Nucleótido Simple , Sitios de Empalme de ARN , Grupos Raciales/genética , Secuencia de Bases , Línea Celular Tumoral , Enfermedad/genética , Exones , Genética de Población , Humanos , Datos de Secuencia Molecular , Mutación , Empalme del ARNRESUMEN
Nucleic acid fragment analysis via separation and detection are routine operations in molecular biology. However, analysis of small single-stranded nucleic acid fragments (<100nt) is challenging and mainly limited to labor-intensive polyacrylamide gel electrophoresis or high-cost capillary electrophoresis methods. Here we report an alternative method, a microfluidic chip electrophoresis system that provides a size resolution of 5nt and a detection time of one minute per sample of fluorescence-labeled DNA/RNA fragments. The feasibility of this system was evaluated by quantifying CRISPR-Cas9 cleavage efficiency and the detection resolution was evaluated by analyzing ssDNA/RNA adenylation and phosphorylation. We employed this system to study the RNA capping efficiency and double-stranded DNA unwinding efficiency in isothermal amplification as two examples for assay design and evaluation. The microfluidic chip electrophoresis system provides a rapid, sensitive, and high-throughput fluorescence fragment analysis (FFA), and can be applied for enzyme characterization, reaction optimization, and product quality control in various molecular biology processes.
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BACKGROUND: The placenta is a key component in understanding the physiological processes involved in pregnancy. Characterizing genes critical for placental function can serve as a basis for identifying mechanisms underlying both normal and pathologic pregnancies. Detailing the placental tissue transcriptome could provide a valuable resource for genomic studies related to placental disease. RESULTS: We have conducted a deep RNA sequencing (RNA-Seq) study on three tissue components (amnion, chorion, and decidua) of 5 human placentas from normal term pregnancies. We compared the placental RNA-Seq data to that of 16 other human tissues and observed a wide spectrum of transcriptome differences both between placenta and other human tissues and between distinct compartments of the placenta. Exon-level analysis of the RNA-Seq data revealed a large number of exons with differential splicing activities between placenta and other tissues, and 79% (27 out of 34) of the events selected for RT-PCR test were validated. The master splicing regulator ESRP1 is expressed at a proportionately higher level in amnion compared to all other analyzed human tissues, and there is a significant enrichment of ESRP1-regulated exons with tissue-specific splicing activities in amnion. This suggests an important role of alternative splicing in regulating gene function and activity in specific placental compartments. Importantly, genes with differential expression or splicing in the placenta are significantly enriched for genes implicated in placental abnormalities and preterm birth. In addition, we identified 604-1007 novel transcripts and 494-585 novel exons expressed in each of the three placental compartments. CONCLUSIONS: Our data demonstrate unique aspects of gene expression and splicing in placental tissues that provide a basis for disease investigation related to disruption of these mechanisms. These data are publicly available providing the community with a rich resource for placental physiology and disease-related studies.
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Perfilación de la Expresión Génica , Placenta/metabolismo , Amnios/metabolismo , Corion/metabolismo , Decidua/metabolismo , Exones/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Especificidad de Órganos , Embarazo , Complicaciones del Embarazo/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARNRESUMEN
Context/objective: At present, there is no consensus on the most effective surgical method for treating symptomatic lumbar spinal stenosis (LSS). Total laminectomy, which is frequently used at this time, destroys the posterior midline structure, causing many postoperative complications. We have designed a new surgical approach instead of total laminectomy. In this paper, we aimed to describe the surgical method of endoscopic modified total laminectomy for lumbar spinal stenosis as well as to explore its early efficacy.Participants: Patients with symptomatic LSS who underwent endoscopic modified total laminoplasty between August 2016 and August 2017 were eligible for our study.Outcome measures: Before surgery and one year after surgery, we measured lower limb pain and back pain by visual analog scale (VAS), disability via Oswestry Disability Index (ODI), and severity of back pain according to the Japanese Orthopedic Association Score for Back Pain (JOA), while any complications were also assessed.Results: Endoscopic modified total laminoplasty was performed on 22 LSS patients, including eight males and 14 females(mean age = 59.3 ± 9.6 years). We found statistically significant differences before and one year after surgery for VAS lower limb pain and back pain, ODI and JOA scores(P < 0.001). Complications included intraoperative dural tears(n = 1),and weak fusion between the lamina and the vertebral body (n = 1).Conclusion: Endoscopic modified total laminectomy is a promising surgical approach which reduces patient suffering and improves patient quality of life.
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Laminoplastia , Traumatismos de la Médula Espinal , Fusión Vertebral , Estenosis Espinal , Anciano , Dolor de Espalda , Descompresión Quirúrgica/métodos , Femenino , Humanos , Laminectomía/efectos adversos , Laminectomía/métodos , Laminoplastia/efectos adversos , Laminoplastia/métodos , Vértebras Lumbares/cirugía , Masculino , Persona de Mediana Edad , Calidad de Vida , Estudios Retrospectivos , Traumatismos de la Médula Espinal/complicaciones , Estenosis Espinal/complicaciones , Estenosis Espinal/cirugía , Resultado del TratamientoRESUMEN
Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. We developed a molecular diagnostic test for SARS-CoV-2, FIND (Fast Isothermal Nucleic acid Detection), based on an enhanced isothermal recombinase polymerase amplification reaction. FIND has a detection limit on patient samples close to that of RT-qPCR, requires minimal instrumentation, and is highly scalable and cheap. It can be performed in high throughput, does not cross-react with other common coronaviruses, avoids bottlenecks caused by the current worldwide shortage of RNA isolation kits, and takes ~45 minutes from sample collection to results. FIND can be adapted to future novel viruses in days once sequence is available. ONE SENTENCE SUMMARY: Sensitive, specific, rapid, scalable, enhanced isothermal amplification method for detecting SARS-CoV-2 from patient samples.
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Rapid, inexpensive, robust diagnostics are essential to control the spread of infectious diseases. Current state of the art diagnostics are highly sensitive and specific, but slow, and require expensive equipment. Here we report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. eRPA does not cross-react with other common coronaviruses, does not require RNA purification, and takes ~45 min from sample collection to results. eRPA represents a first step toward at-home SARS-CoV-2 detection and can be adapted to future viruses within days of genomic sequence availability.
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Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Humanos , ARN/metabolismo , ARN Viral/genética , ARN Viral/aislamiento & purificación , ADN Polimerasa Dirigida por ARN/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinasas/metabolismo , SARS-CoV-2 , Saliva/virología , Virión/genéticaRESUMEN
Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment. This system solves the key challenges in object provenance: persistence in the environment, scalability, rapid and facile decoding, and biocontainment. Our system is compatible with SHERLOCK, a Cas13a RNA-guided nucleic acid detection assay, facilitating its implementation in a wide range of applications.
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Código de Barras del ADN Taxonómico/métodos , ADN Bacteriano/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Microbiología Ambiental , Microbiota/genética , Esporas/genética , Sistemas CRISPR-Cas , ADN Bacteriano/genética , ADN de Hongos/genética , ARN Guía de KinetoplastidaRESUMEN
Here, we report the first example of a hydrogel directly assembled from a copper MOP by a facile ultrasonic procedure, and it exhibited excellent antibacterial activity towards Gram-negative and Gram-positive bacteria.