Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30.363
Filtrar
1.
PLoS One ; 16(9): e0256877, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34473769

RESUMO

In French Polynesia, the first case of SARS-CoV-2 infection was detected on March 10th, 2020, in a resident returning from France. Between March 28th and July 14th, international air traffic was interrupted and local transmission of SARS-CoV-2 was brought under control, with only 62 cases recorded. The main challenge for reopening the air border without requiring travelers to quarantine on arrival was to limit the risk of re-introducing SARS-CoV-2. Specific measures were implemented, including the obligation for all travelers to have a negative RT-PCR test for SARS-CoV-2 carried out within 3 days before departure, and to perform another RT-PCR testing 4 days after arrival. Because of limitation in available medical staff, travelers were provided a kit allowing self-collection of oral and nasal swabs. In addition to increase our testing capacity, self-collected samples from up to 10 travelers were pooled before RNA extraction and RT-PCR testing. When a pool tested positive, RNA extraction and RT-PCR were performed on each individual sample. We report here the results of COVID-19 surveillance (COV-CHECK PORINETIA) conducted between July 15th, 2020, and February 15th, 2021, in travelers using self-collection and pooling approaches. We tested 5,982 pools comprising 59,490 individual samples, and detected 273 (0.46%) travelers positive for SARS-CoV-2. A mean difference of 1.17 Ct (CI 95% 0.93-1.41) was found between positive individual samples and pools (N = 50), probably related to the volume of samples used for RNA extraction (200 µL versus 50 µL, respectively). Retrospective testing of positive samples self-collected from October 20th, 2020, using variants-specific amplification kit and spike gene sequencing, found at least 6 residents infected by the Alpha variant. Self-collection and pooling approaches allowed large-scale screening for SARS-CoV-2 using less human, material and financial resources. Moreover, this strategy allowed detecting the introduction of SARS-CoV-2 variants of concern in French Polynesia.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Programas de Rastreamento/métodos , Vigilância da População/métodos , Manejo de Espécimes/métodos , Viagem , COVID-19/epidemiologia , COVID-19/virologia , Teste para COVID-19/instrumentação , Epidemias/prevenção & controle , França/epidemiologia , Humanos , Polinésia/epidemiologia , Estudos Prospectivos , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Manejo de Espécimes/instrumentação
2.
Front Cell Infect Microbiol ; 11: 691538, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34485174

RESUMO

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, evaluating a proposed workflow amongst the local population is recommended. Here, we aim to validate the collection and treatment of human saliva as a direct specimen for RT-qPCR-based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimens and remained stable for five days either refrigerated or stored at room temperature. The method of processing saliva specimens described in this report bypasses the need for an RNA-extraction process, thereby reducing the cost, time, and manpower required for processing samples. The developed method was tested across nine commercial kits, including the benchmark, to demonstrate its wide applicability on multiple existing workflows. Our developed method achieved an 86% overall agreement rate compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a saliva sampling device, the collection was found to be more convenient for individuals and improved the overall agreement rate to 97%.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Indonésia , Nasofaringe , RNA Viral/genética , Saliva , Manejo de Espécimes
3.
Sci Rep ; 11(1): 17422, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465838

RESUMO

The SARS-CoV-2 pandemic has raised concerns in the identification of the hosts of the virus since the early stages of the outbreak. To address this problem, we proposed a deep learning method, DeepHoF, based on extracting viral genomic features automatically, to predict the host likelihood scores on five host types, including plant, germ, invertebrate, non-human vertebrate and human, for novel viruses. DeepHoF made up for the lack of an accurate tool, reaching a satisfactory AUC of 0.975 in the five-classification, and could make a reliable prediction for the novel viruses without close neighbors in phylogeny. Additionally, to fill the gap in the efficient inference of host species for SARS-CoV-2 using existing tools, we conducted a deep analysis on the host likelihood profile calculated by DeepHoF. Using the isolates sequenced in the earliest stage of the COVID-19 pandemic, we inferred that minks, bats, dogs and cats were potential hosts of SARS-CoV-2, while minks might be one of the most noteworthy hosts. Several genes of SARS-CoV-2 demonstrated their significance in determining the host range. Furthermore, a large-scale genome analysis, based on DeepHoF's computation for the later pandemic in 2020, disclosed the uniformity of host range among SARS-CoV-2 samples and the strong association of SARS-CoV-2 between humans and minks.


Assuntos
COVID-19/virologia , Gatos/virologia , Quirópteros/virologia , Cães/virologia , Vison/virologia , SARS-CoV-2/classificação , Algoritmos , Animais , COVID-19/transmissão , Aprendizado Profundo , Especificidade de Hospedeiro , Humanos , RNA Viral/genética , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Análise de Sequência de RNA
4.
Zhonghua Gan Zang Bing Za Zhi ; 29(8): 803-806, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34517465

RESUMO

Nucleic acid testing is the gold standard for diagnosing HCV infection, and it is of great significance to guide the treatment of HCV patients and change the follow-up strategy. In recent years, its detection technology has become increasingly mature. This article summarizes the necessity and current status of nucleic acid testing for hepatitis C through the analysis of domestic and foreign literature, in order to provide reference for the formulation of relevant policies to eliminate hepatitis C in China.


Assuntos
Hepatite C , RNA Viral , China , Hepacivirus/genética , Hepatite C/diagnóstico , Humanos , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética
5.
Virologie (Montrouge) ; 25(4): 224-235, 2021 08 01.
Artigo em Francês | MEDLINE | ID: mdl-34468319

RESUMO

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.


Assuntos
Vírus de RNA de Cadeia Positiva , Recombinação Genética , Sequência de Bases , RNA Viral/genética , Recombinação Genética/genética , Replicação Viral/genética
6.
Virologie (Montrouge) ; 25(4): 62-73, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34468320

RESUMO

Genetic recombination is a major force driving the evolution of some species of positive sense RNA viruses. Recombination events occur when at least two viruses simultaneously infect the same cell, thereby giving rise to new genomes comprised of genetic sequences originating from the parental genomes. The main mechanism by which recombination occurs involves the viral polymerase that generates a chimera as it switches templates during viral replication. Various experimental systems have alluded to the existence of recombination events that are independent of viral polymerase activity. The origins and the frequency of such events remain to be elucidated to this day. Furthermore, it is not known whether non-replicative recombination yields products that are different from recombinants generated by the viral polymerase. If this is the case, then non-replicative recombination may play a unique role in the evolution of positive sense RNA viruses. Finally, the sparse data available suggest that non-replicative recombination does not necessarily involve only virus-specific sequences. It is thus possible that the non-replicative recombination observed in virus-focused studies may in fact reveal a more generalized mechanism that is non-specific to virus RNAs.


Assuntos
Vírus de RNA de Cadeia Positiva , Recombinação Genética , Sequência de Bases , RNA Viral/genética , Recombinação Genética/genética , Replicação Viral/genética
7.
Anal Chim Acta ; 1180: 338862, 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34538316

RESUMO

Rapid emergence of new strains of drug-resistant H1N1 influenza viruses calls for effective drugs for the controls prior to their outbreaks. In the present work, electrochemical H1N1 RNA beacons have been newly designed for exploring the potentiality of an anticancer agent of Bleomycin (BLM) with Fe (ΙΙ) ions (BLM-Fe(ΙΙ)) alternatively the treatment of drug-resistant H1N1 strains with H274Y gene mutation. Herein, biotinylated (-) ssRNA of H1N1 virus and its complementary (+) ssRNA were labeled with electrochemical signal probes of ferrocene and anthraquinone, respectively. The resultants were hybridized and conjugated with avidin-modified magnetic beads to create electrochemical RNA beacons. The electrochemical signal variation of the H1N1 RNA beacon treated with the RNA degradation agent of BLM-Fe(ΙΙ) were monitored. Results indicate that the BLM-Fe(ΙΙ) agent could effectively cleave both H1N1 dsRNAs and ssRNAs at selective cutting sites, as evidenced by the mass spectrometry analysis. This indicates that the BLM-Fe(II) agent could be utilized to block the viral-host infection process by curbing the host-cell viral RNA-mRNA transcription or inactivate the viruses through the cleavage of viral genomes. The efficiency of the BLM-Fe(ΙΙ) agent was verified with clinical seasonal H1N1 samples using real-time polymerase chain reaction. The therapeutic gene drug of BLM-Fe(ΙΙ) holds great potential for controlling new strains of H1N1 virus resistant to clinical antiviral drugs. More importantly, the so designed RNA beacons may provide a rapid, sensitive and cost-effective platform of drug screening by monitoring the drug-DNA/RNA interactions.


Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Preparações Farmacêuticas , Bleomicina , Farmacorresistência Viral/genética , Compostos Ferrosos , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Neuraminidase , Oseltamivir , RNA Viral/genética
8.
Anal Chim Acta ; 1180: 338846, 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34538333

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a zoonotic RNA virus characterized by high transmission rates and pathogenicity worldwide. Continued control of the COVID-19 pandemic requires the diversification of rapid, easy to use, sensitive, and portable methods for SARS-CoV-2 sample preparation and analysis. Here, we propose a method for SARS-CoV-2 viral enrichment and enzymatic extraction of RNA from clinically relevant matrices in under 10 min. This technique utilizes affinity-capture hydrogel particles to concentrate SARS-CoV-2 from solution, and leverages existing PDQeX technology for RNA isolation. Characterization of our method is accomplished with reverse transcription real-time polymerase chain reaction (RT-PCR) for relative, comparative RNA detection. In a double-blind study analyzing viral transport media (VTM) obtained from clinical nasopharyngeal swabs, our sample preparation method demonstrated both comparable results to a routinely used commercial extraction kit and 100% concordance with laboratory diagnoses. Compatibility of eluates with alternative forms of analysis was confirmed using microfluidic RT-PCR (µRT-PCR), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). The alternative methods explored here conveyed successful amplification from all RNA eluates originating from positive clinical samples. Finally, this method demonstrated high performance within a saliva matrix across a broad range of viral titers and dilutions up to 90% saliva matrix, and sets the stage for miniaturization to the microscale.


Assuntos
COVID-19 , Pandemias , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , SARS-CoV-2
9.
F1000Res ; 10: 373, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34367617

RESUMO

Background: Faecal transplantation is an evidence-based treatment for Clostridioides difficile. Patients infected with SARS-CoV-2 have been shown to shed the virus in stool for up to 33 days, well beyond the average clearance time for upper respiratory tract shedding. We carried out an analytical and clinical validation of reverse-transcriptase quantitative (RT-qPCR) as well as LAMP, LamPORE and droplet digital PCR in the detection of SARS-CoV-2 RNA in stool from donated samples for faecal microbiota transplantation (FMT), spiked samples and asymptomatic inpatients in an acute surgical unit.  Methods: Killed SARS-CoV-2 viral lysate and extracted RNA was spiked into donor stool & FMT and a linear dilution series from 10 -1 to 10 -5 and tested via RT-qPCR, LAMP, LamPORE and ddPCR against SARS-CoV-2. Patients admitted to the critical care unit with symptomatic SARS-CoV-2 and sequential asymptomatic patients from acute presentation to an acute surgical unit were also tested. Results: In a linear dilution series, detection of the lowest dilution series was found to be 8 copies per microlitre of sample. Spiked lysate samples down to 10 -2 dilution were detected in FMT samples using RTQPCR, LamPORE and ddPCR and down to 10 -1 with LAMP. In symptomatic patients 5/12 had detectable SARS-CoV-2 in stool via RT-qPCR and 6/12 via LamPORE, and in 1/97 asymptomatic patients via RT-qPCR. Conclusion: RT-qPCR can be detected in FMT donor samples using RT-qPCR, LamPORE and ddPCR to low levels using validated pathways. As previously demonstrated, nearly half of symptomatic and less than one percent of asymptomatic patients had detectable SARS-CoV-2 in stool.


Assuntos
COVID-19 , SARS-CoV-2 , Transplante de Microbiota Fecal , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real
10.
Sci Rep ; 11(1): 15869, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354202

RESUMO

Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants' occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants' tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants' emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant's tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant's tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/genética , Análise de Sequência de DNA/métodos , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Teste de Ácido Nucleico para COVID-19/métodos , Genoma Viral , Humanos , Nanoporos , RNA Viral/genética , Análise de Sequência de DNA/instrumentação , Glicoproteína da Espícula de Coronavírus/genética
11.
Nat Commun ; 12(1): 4749, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362921

RESUMO

The RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5' end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5' end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Conformação de Ácido Nucleico , RNA Viral/genética , Ribossomos/fisiologia , SARS-CoV-2/genética , COVID-19 , Mutação da Fase de Leitura/genética , Humanos , Pinças Ópticas , RNA Mensageiro/genética
12.
Anal Chem ; 93(33): 11379-11387, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34378378

RESUMO

This work presents a sensitive and specific single-step RNA sensor for Zika virus (ZIKV) in serum. Using AC electrokinetics (ACEK)-enhanced capacitive sensing technology, ZIKV genomic RNA (gRNA) can be directly detected from serum. The sensors are interdigitated electrodes modified with oligonucleotide probes complementary to the conserved regions of ZIKV gRNA. The ACEK capacitive sensing applies an optimized AC excitation signal over the sensor, which induces ACEK microfluidic enrichment of analytes and also simultaneously performs real-time monitoring of hybridization of ZIKV gRNA on the sensor surface. Hence, the sensing procedures are simple with rapid turn-around time and good specificity and sensitivity. A series of experiments are conducted to optimize the sensor performance. The performance of the sensor is investigated for three different probes, two functionalization buffers, and different hybridization buffers. With the optimized sensing protocol, this method can detect spiked ZIKV gRNA from human serum within 30 s and reach a limit of detection of 78.8 copies/µL in analytical samples and as low as 287.5 copies/µL in neat serum. The sensors can successfully differentiate between the RNAs of the ZIKV and dengue virus, two viruses with similar transmission paths and symptoms. The sensor is simple to use and requires no labeling or sophisticated process typically involved in a polymerase chain reaction, hybridization chain reaction, or nucleic acid sequence-based amplification.


Assuntos
Infecção por Zika virus , Zika virus , Genômica , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/genética , Sensibilidade e Especificidade , Zika virus/genética , Infecção por Zika virus/diagnóstico
13.
Viruses ; 13(8)2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34452319

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people globally since its first detection in late 2019. Besides humans, cats and, to some extent, dogs were shown to be susceptible to SARS-CoV-2, highlighting the need for surveillance in a One Health context. Seven veterinary clinics from regions with high incidences of coronavirus disease (COVID-19) were recruited during the early pandemic (March to July 2020) for the screening of patients. A total of 2257 oropharyngeal and nasal swab specimen from 877 dogs and 260 cats (including 18 animals from COVID-19-affected households and 92 animals with signs of respiratory disease) were analyzed for the presence of SARS-CoV-2 RNA using reverse transcriptase real-time polymerase chain reaction (RT-qPCR) targeting the viral envelope (E) and RNA dependent RNA polymerase (RdRp) genes. One oropharyngeal swab from an Italian cat, living in a COVID-19-affected household in Piedmont, tested positive in RT-qPCR (1/260; 0.38%, 95% CI: 0.01-2.1%), and SARS-CoV-2 infection of the animal was serologically confirmed six months later. One oropharyngeal swab from a dog was potentially positive (1/877; 0.1%, 95% CI: 0.002-0.63%), but the result was not confirmed in a reference laboratory. Analyses of convenience sera from 118 animals identified one dog (1/94; 1.1%; 95% CI: 0.02-5.7%) from Lombardy, but no cats (0/24), as positive for anti-SARS-CoV-2 receptor binding domain (RBD) antibodies and neutralizing activity. These findings support the hypothesis that the prevalence of SARS-CoV-2 infection in pet cat and dog populations, and hence, the risk of zoonotic transmission to veterinary staff, was low during the first wave of the pandemic, even in hotspot areas.


Assuntos
COVID-19/veterinária , Doenças do Gato/virologia , Doenças do Cão/virologia , SARS-CoV-2/isolamento & purificação , Animais , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/virologia , Doenças do Gato/diagnóstico , Doenças do Gato/epidemiologia , Gatos , Doenças do Cão/diagnóstico , Doenças do Cão/epidemiologia , Cães , Feminino , Alemanha/epidemiologia , Itália/epidemiologia , Masculino , Orofaringe/virologia , Pandemias , RNA Viral/genética , SARS-CoV-2/genética
14.
Viruses ; 13(8)2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34452352

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.


Assuntos
COVID-19/virologia , Metiltransferases/metabolismo , Capuzes de RNA/genética , RNA Viral/genética , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Anticorpos Monoclonais/análise , Humanos , Metiltransferases/análise , Metiltransferases/genética , Transporte Proteico , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/química , SARS-CoV-2/genética , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/análise , Proteínas Virais Reguladoras e Acessórias/genética
15.
Anal Chem ; 93(35): 11956-11964, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34424659

RESUMO

Coronavirus diseases such as the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose serious threats. Portable and accurate nucleic acid detection is still an urgent need to achieve on-site virus screening and timely infection control. Herein, we have developed an on-site, semiautomatic detection system, aiming at simultaneously overcoming the shortcomings suffered by various commercially available assays, such as low accuracy, poor portability, instrument dependency, and labor intensity. Ultrasensitive isothermal amplification [i.e., reverse transcription loop-mediated isothermal amplification (RT-LAMP)] was applied to generate intensified SARS-CoV-2 RNA signals, which were then transduced to portable commercial pregnancy test strips (PTSs) via ultraspecific human chorionic gonadotropin (hCG)-conjugated toehold-mediated strand exchange (TMSE) probes (hCG-P). The entire detection was integrated into a four-channel, palm-size microfluidic device, named the microfluidic point-of-care (POC) diagnosis system based on the PTS (MPSP) detection system. It provides rapid, cost-effective, and sensitive detection, of which the lowest concentration of detection was 0.5 copy/µL of SARS-CoV-2 RNA, regardless of the presence of other similar viruses, even highly similar severe acute respiratory syndrome coronavirus (SARS-CoV). The successful detection of the authentic samples from different resources evaluated the practical application. The commercial PTS provides a colorimetric visible signal, which is instrument- and optimization-free. Therefore, this MPSP system can be immediately used for SARS-CoV-2 emergency detection, and it is worthy of further optimization to achieve full automation and detection for other infectious diseases.


Assuntos
COVID-19 , Testes de Gravidez , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Gravidez , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e Especificidade
16.
Nat Commun ; 12(1): 5089, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429424

RESUMO

The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL-1. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (<30 min), affordable, highly robust at room temperature (>1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , COVID-19/virologia , Teste para COVID-19 , Humanos , RNA Viral/genética , Recombinação Genética
17.
PLoS One ; 16(8): e0255691, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34351998

RESUMO

Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is clinically essential, and is required also to monitor confirmed cases aiming to prevent further spread. Positive real-time PCR results at late time points following initial diagnosis may be clinically misleading as this methodology cannot account for the infection capabilities and the existence of whole genome sequences. In this study, 47 serial respiratory samples were tested by Allplex-nCoV test (Seegene), a triplex of three assays targeting the SARS-CoV-2 RdRP, E and N genes and subsequently assessed by next generation sequencing (NGS). COVID19 patients were tested at an early stage of the disease, when all these viral gene targets were positive, and at an advanced stage, when only the N gene target was positive in the Allplex-nCoV test. The corresponding NGS results showed the presence of complete viral genome copies at both early and advanced stages of the disease, although the total number of mapped sequences was lower in samples from advanced disease stages. We conclude that reduced viral transmission at this late disease stage may result from the low quantities of complete viral sequences and not solely from transcription favoring the N gene.


Assuntos
COVID-19/genética , SARS-CoV-2/genética , Sequenciamento Completo do Genoma/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , Feminino , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/patogenicidade
18.
PLoS One ; 16(8): e0255690, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34351984

RESUMO

Saliva is an attractive specimen type for asymptomatic surveillance of COVID-19 in large populations due to its ease of collection and its demonstrated utility for detecting RNA from SARS-CoV-2. Multiple saliva-based viral detection protocols use a direct-to-RT-qPCR approach that eliminates nucleic acid extraction but can reduce viral RNA detection sensitivity. To improve test sensitivity while maintaining speed, we developed a robotic nucleic acid extraction method for detecting SARS-CoV-2 RNA in saliva samples with high throughput. Using this assay, the Free Asymptomatic Saliva Testing (IGI FAST) research study on the UC Berkeley campus conducted 11,971 tests on supervised self-collected saliva samples and identified rare positive specimens containing SARS-CoV-2 RNA during a time of low infection prevalence. In an attempt to increase testing capacity, we further adapted our robotic extraction assay to process pooled saliva samples. We also benchmarked our assay against nasopharyngeal swab specimens and found saliva methods require further optimization to match this gold standard. Finally, we designed and validated a RT-qPCR test suitable for saliva self-collection. These results establish a robotic extraction-based procedure for rapid PCR-based saliva testing that is suitable for samples from both symptomatic and asymptomatic individuals.


Assuntos
Teste para COVID-19/métodos , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , Adulto , COVID-19/diagnóstico , Feminino , Humanos , Masculino , Programas de Rastreamento/métodos , RNA/genética , RNA/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Robótica/métodos , Saliva/química , Manejo de Espécimes/métodos
19.
Viruses ; 13(7)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34372527

RESUMO

Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most sensitive and specific assay and, therefore, is the "gold standard" diagnostic method for the diagnosis of SARS-CoV-2 infection. The aim of this study was to compare and analyze the detection performance of three different commercially available SARS-CoV-2 nucleic acid detection kits: Sansure Biotech, GeneFinderTM, and TaqPathTM on 354 randomly selected samples from hospitalized COVID-19 patients. All PCR reactions were performed using the same RNA isolates and one real-time PCR machine. The final result of the three evaluated kits was not statistically different (p = 0.107), and also had a strong positive association and high Cohen's κ coefficient. In contrast, the average Ct values that refer to the ORF1ab and N gene amplification were significantly different (p < 0.001 and p < 0.001, respectively), with the lowest obtained by the TaqPathTM for the ORF1ab and by the Sansure Biotech for the N gene. The results show a high similarity in the analytical sensitivities for SARS-CoV-2 detection, which indicates that the diagnostic accuracy of the three assays is comparable. However, the SanSure Biotech kit showed a bit better diagnostic performance. Our findings suggest that the imperative for improvement should address the determination of cut-off Ct values and rapid modification of the primer sets along with the appearance of new variants.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/virologia , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , COVID-19/epidemiologia , Estudos Transversais , Humanos , RNA Viral/genética , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , Sensibilidade e Especificidade , Sérvia/epidemiologia
20.
Sci Rep ; 11(1): 16145, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373516

RESUMO

The genetic element s2m has been acquired through horizontal transfer by many distantly related viruses, including the SARS-related coronaviruses. Here we show that s2m is evolutionarily conserved in these viruses. Though several lineages of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) devoid of the element can be found, these variants seem to have been short lived, indicating that they were less evolutionary fit than their s2m-containing counterparts. On a species-level, however, there do not appear to be any losses and this pattern strongly suggests that the s2m element is essential to virus replication in SARS-CoV-2 and related viruses. Further experiments are needed to elucidate the function of s2m.


Assuntos
Coronaviridae/genética , Sequências Repetitivas Dispersas/genética , RNA Viral/genética , SARS-CoV-2/genética , Replicação Viral/genética , Animais , Sequência de Bases , COVID-19/virologia , Coronaviridae/classificação , Evolução Molecular , Transferência Genética Horizontal , Humanos , Filogenia , SARS-CoV-2/fisiologia , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...