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1.
J Vis Exp ; (169)2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33871452

RESUMO

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.


Assuntos
/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , /isolamento & purificação , Primers do DNA , Humanos , Pandemias , RNA Viral/genética , Transcrição Reversa , Sensibilidade e Especificidade
2.
Mol Med ; 27(1): 30, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771097

RESUMO

BACKGROUND: SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. METHODS: To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. RESULTS: First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. CONCLUSION: This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.


Assuntos
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Testes Imediatos , /virologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Reversa , Sensibilidade e Especificidade , Carga Viral
3.
PLoS One ; 16(3): e0248042, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33657176

RESUMO

A newly identified coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), has spread rapidly from its epicenter in China to more than 150 countries across six continents. In this study, we have designed three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) primer sets to detect the RNA-dependent RNA polymerase (RdRP), Envelope (E) and Nucleocapsid protein (N) genes of SARS CoV-2. For one tube reaction, the detection limits for five combination SARS CoV-2 LAMP primer sets (RdRP/E, RdRP/N, E/N, RdRP/E/N and RdRP/N/Internal control (actin beta)) were evaluated with a clinical nasopharyngeal swab sample. Among the five combination, the RdRP/E and RdRP/N/IC multiplex LAMP assays showed low detection limits. The sensitivity and specificity of the RT-LAMP assay were evaluated and compared to that of the widely used Allplex™ 2019-nCoV Assay (Seegene, Inc., Seoul, South Korea) and PowerChek™ 2019-nCoV Real-time PCR kit (Kogenebiotech, Seoul, South Korea) for 130 clinical samples from 91 SARS CoV-2 patients and 162 NP specimens from individuals with (72) and without (90) viral respiratory infections. The multiplex RdRP (FAM)/N (CY5)/IC (Hex) RT-LAMP assay showed comparable sensitivities (RdRP: 93.85%, N: 94.62% and RdRP/N: 96.92%) to that of the Allplex™ 2019-nCoV Assay (100%) and superior to those of PowerChek™ 2019-nCoV Real-time PCR kit (RdRP: 92.31%, E: 93.85% and RdRP/E: 95.38%).


Assuntos
/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , /genética , /genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Primers do DNA/genética , Humanos , Proteínas do Nucleocapsídeo/genética , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/genética , Sensibilidade e Especificidade
4.
Water Sci Technol ; 83(5): 1103-1107, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33724939

RESUMO

Noroviruses are significant seafood-borne pathogens, commonly associated with the consumption of filter feeding bivalve molluscs. Here, we report the development of a reverse transcription polymerase chain reaction (RT-PCR) method using primers based on the RNA-dependent RNA polymerase gene of norovirus genogroup II (NoV GII). Samples of bivalves were processed for the concentration of virus and extraction of RNA, followed by reverse transcription PCR. A total of 50 molluscan shellfish samples were analyzed, of which 16 samples yielded positive amplifications of norovirus nucleic acid. The PCR method described here, involving a single set of primers, is useful for rapid screening of shellfish for NoV GII.


Assuntos
Bivalves , Norovirus , Animais , Genótipo , Humanos , Norovirus/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Reversa
5.
PLoS Negl Trop Dis ; 15(3): e0009227, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33647020

RESUMO

Since its first emergence in 2012, cases of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) have continued to occur. At the end of January 2020, 2519 laboratory confirmed cases with a case-fatality rate of 34.3% have been reported. Approximately 84% of human cases have been reported in the tropical region of Saudi Arabia. The emergence of MERS-CoV has highlighted need for a rapid and accurate assay to triage patients with a suspected infection in a timely manner because of the lack of an approved vaccine or an effective treatment for MERS-CoV to prevent and control potential outbreaks. In this study, we present two rapid and visual nucleic acid assays that target the MERS-CoV UpE and N genes as a panel that combines reverse transcription recombinase polymerase amplification with a closed vertical flow visualization strip (RT-RPA-VF). This test panel was designed to improve the diagnostic accuracy through dual-target screening after referencing laboratory testing guidance for MERS-CoV. The limit of detection was 1.2×101 copies/µl viral RNA for the UpE assay and 1.2 copies/µl viral RNA for the N assay, with almost consistent with the sensitivity of the RT-qPCR assays. The two assays exhibited no cross-reactivity with multiple CoVs, including the bat severe acute respiratory syndrome related coronavirus (SARSr-CoV), the bat coronavirus HKU4, and the human coronaviruses 229E, OC43, HKU1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the panel does not require sophisticated equipment and provides rapid detection within 30 min. This panel displays good sensitivity and specificity and may be useful to rapidly detect MERS-CoV early during an outbreak and for disease surveillance.


Assuntos
Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Técnicas de Diagnóstico Molecular/métodos , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa , Arábia Saudita/epidemiologia , Sensibilidade e Especificidade , Proteínas não Estruturais Virais/genética
6.
J Vis Exp ; (168)2021 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-33616108

RESUMO

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.


Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Impressão Tridimensional , Transcrição Reversa/genética , /isolamento & purificação , /genética , Recursos em Saúde , Humanos , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos
7.
Anal Chem ; 93(8): 4126-4133, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33570401

RESUMO

The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV-vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.


Assuntos
Sistemas CRISPR-Cas , Colorimetria/métodos , DNA/química , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , /isolamento & purificação , Proteínas de Bactérias , Sequência de Bases , Proteínas Associadas a CRISPR , DNA/genética , Endodesoxirribonucleases , Ouro/química , Humanos , Nanopartículas Metálicas/química , Fosfoproteínas/genética , Poliproteínas/genética , RNA Viral/genética , Transcrição Reversa , Ressonância de Plasmônio de Superfície , Proteínas Virais/genética
8.
Viruses ; 13(2)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33578999

RESUMO

Since the discovery of HIV-1, the viral capsid has been recognized to have an important role as a structural protein that holds the viral genome, together with viral proteins essential for viral life cycle, such as the reverse transcriptase (RT) and the integrase (IN). The reverse transcription process takes place between the cytoplasm and the nucleus of the host cell, thus the Reverse Transcription Complexes (RTCs)/Pre-integration Complexes (PICs) are hosted in intact or partial cores. Early biochemical assays failed to identify the viral CA associated to the RTC/PIC, possibly due to the stringent detergent conditions used to fractionate the cells or to isolate the viral complexes. More recently, it has been observed that some host partners of capsid, such as Nup153 and CPSF6, can only bind multimeric CA proteins organized in hexamers. Those host factors are mainly located in the nuclear compartment, suggesting the entrance of the viral CA as multimeric structure inside the nucleus. Recent data show CA complexes within the nucleus having a different morphology from the cytoplasmic ones, clearly highlighting the remodeling of the viral cores during nuclear translocation. Thus, the multimeric CA complexes lead the viral genome into the host nuclear compartment, piloting the intranuclear journey of HIV-1 in order to successfully replicate. The aim of this review is to discuss and analyze the main discoveries to date that uncover the viral capsid as a key player in the reverse transcription and PIC maturation until the viral DNA integration into the host genome.


Assuntos
Capsídeo/metabolismo , Núcleo Celular/virologia , HIV-1/fisiologia , Transporte Ativo do Núcleo Celular , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , HIV-1/química , HIV-1/metabolismo , Modelos Biológicos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transcrição Reversa , Integração Viral , Replicação Viral
9.
Front Cell Infect Microbiol ; 11: 613304, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33598439

RESUMO

Background: The emerging Coronavirus Disease-2019 (COVID-19) has challenged the public health globally. With the increasing requirement of detection for SARS-CoV-2 outside of the laboratory setting, a rapid and precise Point of Care Test (POCT) is urgently needed. Methods: Targeting the nucleocapsid (N) gene of SARS-CoV-2, specific primers, and probes for reverse transcription recombinase-aided amplification coupled with lateral flow dipstick (RT-RAA/LFD) platform were designed. For specificity evaluation, it was tested with human coronaviruses, human influenza A virus, influenza B viruses, respiratory syncytial virus, and hepatitis B virus, respectively. For sensitivity assay, it was estimated by templates of recombinant plasmid and pseudovirus of SARS-CoV-2 RNA. For clinical assessment, 100 clinical samples (13 positive and 87 negatives for SARS-CoV-2) were tested via quantitative reverse transcription PCR (RT-qPCR) and RT-RAA/LFD, respectively. Results: The limit of detection was 1 copies/µl in RT-RAA/LFD assay, which could be conducted within 30 min at 39°C, without any cross-reaction with other human coronaviruses and clinical respiratory pathogens. Compared with RT-qPCR, the established POCT assay offered 100% specificity and 100% sensitivity in the detection of clinical samples. Conclusion: This work provides a convenient POCT tool for rapid screening, diagnosis, and monitoring of suspected patients in SARS-CoV-2 endemic areas.


Assuntos
/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , /genética , /virologia , /genética , Primers do DNA/genética , Humanos , Fosfoproteínas/genética , Testes Imediatos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Recombinases/metabolismo , Transcrição Reversa , Sensibilidade e Especificidade
10.
J Transl Med ; 19(1): 74, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33593370

RESUMO

BACKGROUND: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. METHODS: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19. RESULTS: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input. CONCLUSIONS: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.


Assuntos
/métodos , /virologia , Sistemas CRISPR-Cas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa/genética , /isolamento & purificação , Sequência de Bases , Humanos , Limite de Detecção , RNA Viral/genética , Padrões de Referência , /genética
11.
Biotechniques ; 70(3): 167-174, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33535813

RESUMO

The ongoing pandemic has demonstrated the utility of widespread surveillance and diagnostic detection of the novel SARS-CoV-2. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) has enabled broader testing, but current LAMP tests only detect single targets and require separate reactions for controls. With flu season in the Northern Hemisphere, the ability to screen for multiple targets will be increasingly important, and the ability to include internal controls in RT-LAMP allows for improved efficiency. Here we describe multiplexed RT-LAMP with four targets (SARS-CoV-2, influenza A, influenza B, human RNA) in a single reaction using real-time and end point fluorescence detection. Such increased functionality of RT-LAMP will enable even broader adoption of this molecular testing approach and aid in the fight against this public health threat.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza B/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , /genética , /métodos , Primers do DNA/genética , Fluorescência , RNA Viral/análise , Transcrição Reversa , Sensibilidade e Especificidade
12.
Int J Infect Dis ; 104: 303-305, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33434671

RESUMO

BACKGROUND: Multiple molecular kits are available for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) worldwide, with many lacking proper clinical evaluation due to the emergency caused by the coronavirus disease 2019 (COVID-19) pandemic, particularly in developing countries. METHODS: This study was conducted to evaluate the clinical performance of the Isopollo COVID-19 detection kit (M Monitor, South Korea) for reverse transcription loop-mediated isothermal amplification (RT-LAMP) SARS-CoV-2 diagnosis, using the SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) protocol as the gold standard. RESULTS: A total of 220 clinical samples were included in the study; 168 samples were SARS-CoV-2-positive and 52 samples were SARS-CoV-2-negative according to the SARS-CoV-2 RT-PCR protocol. For the Isopollo COVID-19 detection kit, only 104 out of 168 samples were SARS-CoV-2-positive. This result shows a low clinical performance, with sensitivity of 61.9% for the evaluated RT-LAMP assay. CONCLUSIONS: Proper clinical performance evaluation studies by regulatory agencies in developing countries such as Ecuador should be mandatory prior to clinical use authorization of SARS-CoV-2 diagnosis kits, particularly when those kits lack either US Food and Drug Administration or country of origin clinical use authorization.


Assuntos
/diagnóstico , Pandemias , Kit de Reagentes para Diagnóstico/normas , /isolamento & purificação , /epidemiologia , Países em Desenvolvimento , Equador/epidemiologia , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Transcrição Reversa , /genética
13.
PLoS One ; 16(1): e0245164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33406112

RESUMO

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/µL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.


Assuntos
/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , /genética , /genética , Humanos , Malásia/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/genética , Recombinases/metabolismo , Transcrição Reversa/genética , Sensibilidade e Especificidade
14.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33498408

RESUMO

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.


Assuntos
/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas Virais/genética , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Poliproteínas/genética , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcrição Reversa
15.
Mol Genet Genomics ; 296(2): 409-422, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33464395

RESUMO

RNA debranching enzymes are 2'-5' phosphodiesterases found in all eukaryotes. Their main role is cleavage of intron RNA lariat branch points, promoting RNA turnover via exonucleases. Consistent with this role, cells with reduced RNA debranching enzyme activity accumulate intron RNA lariats. The Saccharomyces cerevisiae RNA debranching enzyme Dbr1p is also a host factor for the yeast long terminal repeat (LTR) retrotransposon Ty1, a model for many aspects of retroviral replication. Fittingly, the human RNA debranching enzyme Dbr1 is a host factor for the human immunodeficiency virus, HIV-1. The yeast and human RNA debranching enzymes act at the reverse transcription stages for Ty1 and HIV-1, respectively. Although efficient production of full-length Ty1 cDNA requires Dbr1p, the findings reported here indicate that production of the earliest distinct cDNA product, minus strand strong stop DNA (-sssDNA), is equivalent in wild type and dbr1∆ mutant cells. Several branched Ty1 RNAs are shown to accumulate in dbr1∆ cells during retrotransposition. These data are consistent with creation of Ty1 RNA branches prior to Ty1 reverse transcription and their removal by Dbr1p to allow efficient extension of early cDNA products. The data support the possibility that RNA branch formation and cleavage play broadly shared, but unknown roles in retroviral and LTR retrotransposon reverse transcription.


Assuntos
RNA Nucleotidiltransferases/genética , Transcrição Reversa , Saccharomyces cerevisiae/enzimologia , DNA de Cadeia Simples/metabolismo , HIV-1/genética , Mutação , RNA Nucleotidiltransferases/metabolismo , Retroelementos , Saccharomyces cerevisiae/genética , Replicação Viral
16.
Sci Rep ; 11(1): 2402, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510181

RESUMO

The COVID-19 pandemic has resulted in an urgent need for a rapid, point of care diagnostic testing that could be rapidly scaled on a worldwide level. We developed and tested a highly sensitive and robust assay based on reverse transcription loop mediated isothermal amplification (RT-LAMP) that uses readily available reagents and a simple heat block using contrived spike-in and actual clinical samples. RT-LAMP testing on RNA-spiked samples showed a limit of detection (LoD) of 2.5 copies/µl of viral transport media. RT-LAMP testing directly on clinical nasopharyngeal swab samples in viral transport media had an 85% positive percentage agreement (PPA) (17/20), and 100% negative percentage agreement (NPV) and delivered results in 30 min. Our optimized RT-LAMP based testing method is a scalable system that is sufficiently sensitive and robust to test for SARS-CoV-2 directly on clinical nasopharyngeal swab samples in viral transport media in 30 min at the point of care without the need for specialized or proprietary equipment or reagents. This cost-effective and efficient one-step testing method can be readily available for COVID-19 testing world-wide, especially in resource poor settings.


Assuntos
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , /genética , Técnicas de Laboratório Clínico/métodos , Técnicas e Procedimentos Diagnósticos , Testes Diagnósticos de Rotina , Humanos , Limite de Detecção , Testes Imediatos , RNA Viral/genética , Transcrição Reversa , /metabolismo , Sensibilidade e Especificidade
17.
Methods Mol Biol ; 2167: 147-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712919

RESUMO

Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.


Assuntos
Proteínas Arqueais/química , Pegada de DNA/métodos , Ácido Edético/análogos & derivados , Radical Hidroxila/química , Dobramento de RNA/genética , RNA/química , Ribonuclease P/metabolismo , Sítios de Ligação , Ácido Edético/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica , RNA Catalítico/genética , RNA Catalítico/metabolismo , Transcrição Reversa , Ribonuclease P/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Análise de Sequência de DNA
18.
Methods Mol Biol ; 2167: 171-182, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712920

RESUMO

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a widely used technique for studying the structure and function of RNA molecules. It characterizes the flexibility of single nucleotides in the context of the local RNA structure. Here we describe the application of SHAPE-MaP (mutational profiling) to study different conformational states of the group II intron during the self-splicing reaction.


Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Íntrons/genética , Conformação de Ácido Nucleico , Processamento de RNA/genética , RNA/química , Análise de Sequência de RNA/métodos , Acilação , Mutação , RNA/genética , Transcrição Reversa , Software
19.
Methods Mol Biol ; 2209: 163-173, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201469

RESUMO

Multiple different methods have been employed to investigate the unwinding of RNA G-quadruplexes by various helicase proteins. Each has their own pitfalls, namely, looking at non-native or chemically modified RNA sequences, biasing the unwinding process with competing trap nucleotides, and a lack of context sequence to the 5' and 3' of the RNA G-quadruplex structure. Herein we present two straightforward methods that allow for quadruplex unwinding to be monitored on native RNA sequences without the use of fluorescent modifications, specialized equipment, or trap nucleotides to be employed.


Assuntos
RNA Helicases DEAD-box/química , Quadruplex G , RNA/química , Transcrição Reversa , Humanos , Conformação de Ácido Nucleico , Proteínas Recombinantes/química
20.
Exp Parasitol ; 222: 108062, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33383024

RESUMO

Long non-coding RNAs (lncRNAs) perform several types of regulatory functions and have been recently explored in the genus Schistosoma. Although sequencing and bioinformatics approaches have demonstrated the presence of hundreds of lncRNAs and microRNAs (miRNAs) in this genus, information regarding their abundance, characteristics, and potential functions linked to Schistosoma mansoni biology and parasite-host interaction is limited. Our objectives in the present study were to verify whether 15 previously identified S. mansoni lncRNAs are detectable in the host liver. In addition, we assess whether these lncRNAs are present in the S. mansoni infective form and the stages inside the definitive host. The detection of these 15 S. mansoni lncRNAs and a long terminal repeat (LTR) retrotransposon Saci 4 was performed in the eggs, cercariae, and 3.5-h schistosomula. All lncRNAs were found to be expressed in these stages; some of the lncRNAs were found in the livers of the infected C57BL/6 mice. In conclusion, S. mansoni lncRNAs were detected in host livers and quantified. Furthermore, many of the lncRNAs analyzed showed differential expression in the larval stages, indicating that they play a stage-specific regulatory role.


Assuntos
Fígado/parasitologia , RNA Longo não Codificante/isolamento & purificação , Schistosoma mansoni/genética , Esquistossomose mansoni/parasitologia , Animais , Mapeamento Cromossômico , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Retroelementos/fisiologia , Transcrição Reversa , Schistosoma mansoni/crescimento & desenvolvimento , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/patologia
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