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1.
J Biomed Nanotechnol ; 17(7): 1364-1370, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34446139

RESUMO

Researchers have conducted in-depth research on DNA methylation mechanism, which is related to various diseases such as deficiency of imprinted gene and occurrence of tumors. This study provides a novel rapid quantitative detection assay and real-time fluorescence recombinase-aided amplification assay (RAA) for DNA methylation. Firstly, specific sequence of methylation genes was chosen and primers and fluorogenic probe for RAA experiment were designed and synthesized. Lastly, these amplification products were proven by sequencing and analysis. Results showed that the amplification efficiency and template concentration of RAA had linear dependent (R² > 95%) when the concentration range was 4.64×108 copies/µL˜4.64×104 copies/µL. The test assay can also detect positive samples when the template concentration is below 4.64×104 copies/µL. Remarkably, the entire experiment process only takes 15-20 minutes, so it is beneficial for rapid bedside simple screening of some special DNA methylation sites, such as detection of resistance genes. In a word, this method has very great potential for diseases with DNA methylation in clinical settings, especially if methylation analysis needs to be done quickly and easily.


Assuntos
Metilação de DNA , Recombinases , Primers do DNA , Técnicas de Amplificação de Ácido Nucleico , Recombinases/genética , Recombinases/metabolismo , Sensibilidade e Especificidade
2.
Yi Chuan ; 43(8): 802-812, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34413019

RESUMO

The genetically modified (GM) maize 'Shuangkang'12-5 has good insect resistance and herbicide tolerance, which is one of the first series of GM maizes obtained a safety certificate in China, and it has broad application prospect in the future. This study established an on-site rapid detection method for GM 'Shuangkang'12-5 based on recombinase polymerase amplification (RPA) technology, which primes and probe were designed according to the specific flank sequence. Then the best combination of primers and probe was obtained through a screeing process. The amplification results of fluorescence RPA can be directly visualized under blue light. The results showed that the visual detection system of GM 'Shuangkang'12-5 with high specificity, and the detection sensitivity of the method could reached 10 copies. Further research found that the RPA amplification system had a wide range of temperature (34℃-46℃). According to this property, the common self-heating warm pastes on the market were used replace the traditional heating instruments to stimulate the RPA.The results showed that the self-heating warm paste meets the temperature requirement of the RPA system. Finally, we combined the self-heating warm pastes with the RPA visual detection system to conduct on-site detection of GM 'Shuangkang'12-5, and compared the results with the detection results of qPCR. The detection showed that the results of on-site visual detection method established in this study were consistent with the detection results of the qPCR. Moreover, the visual detection method was more shorter in time and the final detection result was clear and easy to distinguish. The rapid on-site visual detection method for GM 'Shuangkang' 12-5 established in this study has high specificity, high sensitivity and convenience. It not only meets the needs of on-site rapid detection of GM 'Shuangkang'12-5, but also provides highlight for the development of other on-site rapid detection methods.


Assuntos
Recombinases , Zea mays , Primers do DNA , Técnicas de Amplificação de Ácido Nucleico , Nucleotidiltransferases , Zea mays/genética
3.
Viruses ; 13(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34372564

RESUMO

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.


Assuntos
Bornaviridae/genética , Bornaviridae/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Animais , Doenças das Aves/virologia , Aves/genética , Aves/virologia , Primers do DNA/genética , Genoma Viral , Infecções por Mononegavirales/veterinária , Papagaios/genética , Papagaios/virologia , Passeriformes/genética , Passeriformes/virologia , Filogenia , RNA Viral/genética , Sequenciamento Completo do Genoma/métodos
4.
Genomics ; 113(5): 3174-3184, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34293476

RESUMO

As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver named CoVrimer by which users can search for and align existing or newly designed conserved/degenerate primer pair sequences against the viral genome and assess the mutation load of both primers and amplicons. CoVrimer uses mutation data obtained from an online platform established by NGDC-CNCB (12 May 2021) to identify genomic regions, either conserved or with low levels of mutations, from which potential primer pairs are designed and provided to the user for filtering based on generalized and SARS-CoV-2 specific parameters. Alignments of primers and probes can be visualized with respect to the reference genome, indicating variant details and the level of conservation. Consequently, CoVrimer is likely to help researchers with the challenges posed by viral evolution and is freely available at http://konulabapps.bilkent.edu.tr:3838/CoVrimer/.


Assuntos
Primers do DNA/química , SARS-CoV-2/genética , Análise de Sequência de DNA/métodos , Software , Sequência Conservada , Primers do DNA/genética , Genoma Viral , Mutação
5.
Methods Mol Biol ; 2320: 247-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302663

RESUMO

A knock-in can generate fluorescent or Cre-reporter under the control of an endogenous promoter. It also generates knock-out or tagged-protein with fluorescent protein and short tags for tracking and purification. Recent advances in genome editing with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) significantly increased the efficiencies of making knock-in cells. Here we describe the detailed protocols of generating knock-in mouse and human pluripotent stem cells (PSCs) by electroporation and lipofection, respectively.


Assuntos
Sistemas CRISPR-Cas , Técnicas de Introdução de Genes/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Células Cultivadas , Células Clonais , Meios de Cultura , Primers do DNA , Resistência a Medicamentos/genética , Eletroporação , Células-Tronco Embrionárias/citologia , Edição de Genes/métodos , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Puromicina/farmacologia , RNA Guia/genética , Reparo de DNA por Recombinação/genética
6.
J Virol Methods ; 296: 114227, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34224752

RESUMO

The rapid detection of novel pathogens including SARS-CoV-2 necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Delay in diagnosis has facilitated the rapid spread of this novel virus throughout the world resulting in global mortality that will surpass 2.5 million people. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral cDNA nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 nasopharyngeal samples using CDC's N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100 % concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Primers do DNA , Testes Diagnósticos de Rotina , Humanos , Testes Imediatos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Recombinases/química , SARS-CoV-2/genética , Sensibilidade e Especificidade
7.
Methods Mol Biol ; 2328: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251628

RESUMO

Plants use different regulatory modules in response to changes in their surroundings. With the transcriptomic approaches governing all research areas, an integrative, fast, and sensitive approach toward validating genes of interest becomes a critical step prior to functional studies in planta. This chapter describes a detailed method for a quantitative analysis of transcriptional readouts of defense response genes using tobacco leaves as a transient system. The method uses Luciferase reporter assays to monitor activities of defense pathway promoters. Under normal conditions, the JASMONATE ZIM-DOMAIN (JAZ) proteins repress defense genes by preventing their expression. Here, we will provide a detailed protocol on the use of a dual-luciferase system to analyze activities of various defense response promoters simultaneously. We will use two well-characterized modules from the Jasmonic acid (JA) defense pathway; the JAZ3 repressor protein and the promoters of three of JA responsive genes, MYC2, 3 and 4. This assay revealed not only differences in promoter strength but also provided quantitative insights on the JAZ3 repression of MYCs in a quantitative manner.


Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Proteínas Repressoras/metabolismo , Tabaco/metabolismo , Agrobacterium tumefaciens/metabolismo , Primers do DNA , Genes myc/genética , Luciferases/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Proteínas Repressoras/genética , Tabaco/genética
8.
ACS Appl Mater Interfaces ; 13(26): 30295-30305, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165969

RESUMO

As viruses have been threatening global public health, fast diagnosis has been critical to effective disease management and control. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is now widely used as the gold standard for detecting viruses. Although a multiplex assay is essential for identifying virus types and subtypes, the poor multiplicity of RT-qPCR makes it laborious and time-consuming. In this paper, we describe the development of a multiplex RT-qPCR platform with hydrogel microparticles acting as independent reactors in a single reaction. To build target-specific particles, target-specific primers and probes are integrated into the particles in the form of noncovalent composites with boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs). The thermal release characteristics of DNA, primer, and probe from the composites of primer-BNNT and probe-CNT allow primer and probe to be stored in particles during particle production and to be delivered into the reaction. In addition, BNNT did not absorb but preserved the fluorescent signal, while CNT protected the fluorophore of the probe from the free radicals present during particle production. Bicompartmental primer-incorporated network (bcPIN) particles were designed to harness the distinctive properties of two nanomaterials. The bcPIN particles showed a high RT-qPCR efficiency of over 90% and effective suppression of non-specific reactions. 16-plex RT-qPCR has been achieved simply by recruiting differently coded bcPIN particles for each target. As a proof of concept, multiplex one-step RT-qPCR was successfully demonstrated with a simple reaction protocol.


Assuntos
Hidrogéis/química , Reação em Cadeia da Polimerase Multiplex/métodos , Nanotubos de Carbono/química , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Compostos de Boro/química , Coronavirus/química , Primers do DNA/química , DNA de Cadeia Simples/química , Corantes Fluorescentes/química , Grafite/química , Vírus da Influenza A/química , Vírus da Doença de Newcastle/química , Estudo de Prova de Conceito , RNA Viral/química , Viroses/diagnóstico
9.
Lett Appl Microbiol ; 73(3): 363-371, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34101222

RESUMO

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 103  CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 104  CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.


Assuntos
Fusarium , Micotoxinas , Zearalenona , Primers do DNA , Contaminação de Alimentos/análise , Fusarium/genética , Reação em Cadeia da Polimerase Multiplex
10.
Nat Commun ; 12(1): 3690, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140468

RESUMO

CRISPR-Cas pathways provide prokaryotes with acquired "immunity" against foreign genetic elements, including phages and plasmids. Although many of the proteins associated with CRISPR-Cas mechanisms are characterized, some requisite enzymes remain elusive. Genetic studies have implicated host DNA polymerases in some CRISPR-Cas systems but CRISPR-specific replicases have not yet been discovered. We have identified and characterised a family of CRISPR-Associated Primase-Polymerases (CAPPs) in a range of prokaryotes that are operonically associated with Cas1 and Cas2. CAPPs belong to the Primase-Polymerase (Prim-Pol) superfamily of replicases that operate in various DNA repair and replication pathways that maintain genome stability. Here, we characterise the DNA synthesis activities of bacterial CAPP homologues from Type IIIA and IIIB CRISPR-Cas systems and establish that they possess a range of replicase activities including DNA priming, polymerisation and strand-displacement. We demonstrate that CAPPs operonically-associated partners, Cas1 and Cas2, form a complex that possesses spacer integration activity. We show that CAPPs physically associate with the Cas proteins to form bespoke CRISPR-Cas complexes. Finally, we propose how CAPPs activities, in conjunction with their partners, may function to undertake key roles in CRISPR-Cas adaptation.


Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroidetes/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA Primase/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/genética , Bacteroidetes/enzimologia , Biologia Computacional , DNA Primase/genética , Primers do DNA/biossíntese , DNA Polimerase Dirigida por DNA/genética , Dimerização , Escherichia coli/metabolismo , Expressão Gênica , Mutação , Filogenia , Células Procarióticas/metabolismo , Proteínas Recombinantes , Ribonucleotídeos/metabolismo
11.
BMC Infect Dis ; 21(1): 598, 2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162342

RESUMO

BACKGROUND: Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. METHODS: In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. RESULTS: Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75-91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. CONCLUSIONS: For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50-91.49%) and specificity (89.23-100%), depending on the DNA extraction methods used.


Assuntos
Úlcera de Buruli/diagnóstico , DNA Bacteriano/isolamento & purificação , Técnicas de Diagnóstico Molecular , Mycobacterium ulcerans/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , Úlcera de Buruli/microbiologia , Primers do DNA , Elementos de DNA Transponíveis , Humanos , Mycobacterium ulcerans/genética , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade
12.
Biochem Biophys Res Commun ; 567: 195-200, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34166918

RESUMO

Recombinase polymerase amplification (RPA) is an isothermal reaction that amplifies a target DNA sequence with a recombinase, a single-stranded DNA-binding protein (SSB), and a strand-displacing DNA polymerase. In this study, we optimized the reaction conditions of RPA to detect SARS-CoV-2 DNA and RNA using a statistical method to enhance the sensitivity. In vitro synthesized SARS-CoV-2 DNA and RNA were used as targets. After evaluating the concentration of each component, the uvsY, gp32, and ATP concentrations appeared to be rate-determining factors. In particular, the balance between the binding and dissociation of uvsX and DNA primer was precisely adjusted. Under the optimized condition, 60 copies of the target DNA were specifically detected. Detection of 60 copies of RNA was also achieved. Our results prove the fabrication flexibility of RPA reagents, leading to an expansion of the use of RPA in various fields.


Assuntos
DNA Viral/análise , DNA Polimerase Dirigida por DNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/normas , RNA Viral/análise , Recombinases/metabolismo , SARS-CoV-2/genética , Estatística como Assunto , Primers do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , SARS-CoV-2/isolamento & purificação , Proteínas Virais/metabolismo
13.
Mol Cell Probes ; 58: 101744, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34089805

RESUMO

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes. The overall sensitivity and specificity of the SORP assay was: 99.31% (95% CI: 96.22-99.98%), 100.0% (95% CI: 98.4-100%) respectively. The SORP assay could also detect a panel of variants of concern (VOC) from the B1.1.7 (UK) and B1.351 (SA) lineage. In summary, access to a repertoire of new SARS-CoV-2 LDT's would assist diagnostic laboratories in developing strategies to overcome some of the testing issues encountered during high-throughput SARS-CoV-2 testing.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Laboratório Clínico/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , COVID-19/virologia , Primers do DNA/genética , Sondas de DNA/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Reprodutibilidade dos Testes , Ribonuclease P/genética , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais/genética
14.
Mol Cell Probes ; 58: 101748, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34146663

RESUMO

Covid-19 disease caused by SARS-CoV-2 is still being transmitted in developed and developing countries irrespective of healthcare setups. India with 1.3 billion people in the world is severely affected by Covid-19 with 11.3 million cases and 157 000 deaths so far. We have assessed the mismatches in WHO recommended rRT-PCR assays primer and probe binding regions against SARS-CoV-2 Indian genome sequences through in-silico bioinformatics analysis approach. Primers and probe sequences belonging to CN-CDC-ORF1ab from China and HKU-ORF1b from Hong Kong targeting ORF1ab gene while NIH-TH-N from Thailand, HKU-N from Hong Kong and US-CDCN-2 from USA targeting N genes displayed accurate matches (>98.3%) with the 2019 novel corona virus sequences from India. On the other hand, none of the genomic sequences displayed exact match with the primer/probe sequences belonging to Charité-ORF1b from Germany targeting ORF1ab gene. We think it will be worthwhile to release this information to the clinical and medical communities working in Indian Covid-19 frontline taskforce to tackle the recently emerging Covid-19 outbreaks as of March-2021.


Assuntos
COVID-19/diagnóstico , Simulação por Computador , Genoma Viral/genética , Mutação , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/epidemiologia , COVID-19/virologia , Primers do DNA/genética , Sondas de DNA/genética , Surtos de Doenças , Humanos , Índia/epidemiologia , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , SARS-CoV-2/fisiologia , Sensibilidade e Especificidade
15.
J Virol Methods ; 295: 114215, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34166701

RESUMO

BACKGROUND: This study aimed to evaluate the impact of four different reverse transcription quantitative PCR (RT-qPCR) master mixes on the performance of SARS-CoV-2 diagnostic PCRs using three primer/probe assays targeting the N gene (A, B and C). The dynamic range and lowest detected quantity was determined using a SARS-CoV-2 partial N gene RNA transcript dilution series (100,000-1 copy/µl) and verified using 72 nose and throat swabs, 29 of which tested positive for SARS-CoV-2 RNA. RESULTS: Assay C consistently detected the lowest quantity of partial N gene RNA transcript with all mastermixes. The Takara One Step PrimeScript™ III RT-PCR Kit mastermix enabled all primer pairs to detect the entire dynamic range evaluated, with the Qiagen Quantifast and Thermofisher TaqPath 1-Step kits also performing well. Sequences from all three primer/probe sets tested in this study (assay A, B and C) have 100 % homology to ≥97 % of the of SARS-CoV-2 sequences available up to 31st December 2020 (n = 291,483 sequences). CONCLUSIONS: This work demonstrates that specific assays (in this case assay C) can perform well in terms of dynamic range and lowest detected quantity regardless of the mastermix used. However we also show that, by choosing the most appropriate mastermix, poorer performing primer pairs are also able to detect all of the template dilutions investigated. This work increases the potential options when choosing assays for SARS-CoV-2 diagnosis and provides solutions to enable them to work with optimal analytical sensitivity.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Proteínas do Nucleocapsídeo de Coronavírus/genética , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Teste de Ácido Nucleico para COVID-19/instrumentação , Primers do DNA/genética , Humanos , Nariz/virologia , Faringe/virologia , Fosfoproteínas/genética , RNA Viral/genética , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Sensibilidade e Especificidade , Homologia de Sequência do Ácido Nucleico
16.
Nat Commun ; 12(1): 3518, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112775

RESUMO

DNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent biomolecular structure of DNA which differentiates it from conventional storage media. The unique molecular architecture of DNA storage also prompts important discussions on how data should be organized, accessed, and manipulated and what practical functionalities may be possible. Here we leverage thermodynamic tuning of biomolecular interactions to implement useful data access and organizational features. Specific sets of environmental conditions including distinct DNA concentrations and temperatures were screened for their ability to switchably access either all DNA strands encoding full image files from a GB-sized background database or subsets of those strands encoding low resolution, File Preview, versions. We demonstrate File Preview with four JPEG images and provide an argument for the substantial and practical economic benefit of this generalizable strategy to organize data.


Assuntos
DNA/química , Processamento de Imagem Assistida por Computador/métodos , Armazenamento e Recuperação da Informação/métodos , Simulação por Computador , Primers do DNA/química , Bases de Dados de Ácidos Nucleicos , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase em Tempo Real/métodos , Software , Temperatura , Termodinâmica
17.
Biomed Res Int ; 2021: 6653950, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34124257

RESUMO

The study is aimed at establishing the optimal parameters for RNA purification of pooled specimens, in SARS-CoV-2 assay. This research work evaluates the difference of extracted RNA purity of pooled samples with and without treatment with isopropyl alcohol and its effect on real-time RT-PCR. As per the protocol of the Indian Council of Medical Research (ICMR), 5 sample pools were analysed using qRT-PCR. A total of 100 pooled samples were selected for the study by mixing 50 µL of one COVID-19 positive nasopharyngeal/oropharyngeal (NP/OP) specimen and 50 µL each of 4 known negative specimens. Pool RNA was extracted using the column-based method, and 1 set of pooled extracted RNA was tested as such, while RNA of the second set was treated additionally with chilled isopropyl alcohol (modified protocol). Further, the purity of extracted RNA in both the groups was checked using Microvolume Spectrophotometers (Nanodrop) followed by RT-PCR targeting E-gene and RNaseP target. The results showed that the purity index of extracted RNA of untreated pooled specimens was inferior to isopropyl alcohol-treated templates, which was observed to be 85% sensitivity and 100% specificity. The average Cq (E gene) in the unpurified and purified pool RNA group was 34.66 and 31.48, respectively. The nanodrop data suggested that purified RNA concentration was significantly increased with an average value of 24.73 ± 1.49 ng/uL, which might be the reason for high sensitivity and specificity. Thus, this group testing of SARS-CoV-2 cases using pools of 5 individual samples would be the best alternative for saving molecular reagents, personnel time, and can increase the overall testing capacity. However, purity of RNA is one of the important determinants to procure unfailing results, thus, this additional purification step must be included in the protocol after RNA has been extracted using commercially available kit before performing qRT-PCR.


Assuntos
COVID-19/diagnóstico , Proteínas do Envelope de Coronavírus/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , 2-Propanol/química , Biomarcadores/análise , COVID-19/virologia , Primers do DNA/síntese química , Primers do DNA/genética , Humanos , Nasofaringe/virologia , Orofaringe/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
Sci Rep ; 11(1): 12565, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131209

RESUMO

Accurate designing of polymerase chain reaction (PCR) primers targeting conserved segments in viral genomes is desirable for preventing false-negative results and decreasing the need for standardization across different PCR protocols. In this work, we designed and described a set of primers and probes targeting conserved regions identified from a multiple sequence alignment of 2341 Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) genomes from the Global Initiative on Sharing All Influenza Data (GISAID). We subsequently validated those primers and probes in 211,833 SARS-CoV-2 whole-genome sequences. We obtained nine systems (forward primer + reverse primer + probe) that potentially anneal to highly conserved regions of the virus genome from these analyses. In silico predictions also demonstrated that those primers do not bind to nonspecific targets for human, bacterial, fungal, apicomplexan, and other Betacoronaviruses and less pathogenic sub-strains of coronavirus. The availability of these primer and probe sequences will make it possible to validate more efficient protocols for identifying SARS-CoV-2.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Reação em Cadeia da Polimerase/métodos , SARS-CoV-2/genética , Simulação por Computador , Primers do DNA , Genoma Viral , Humanos , SARS-CoV-2/isolamento & purificação , Sequenciamento Completo do Genoma
19.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070906

RESUMO

A rapid and accurate PCR-based method was developed in this study for detecting and identifying a new species of root-lesion nematode (Pratylenchus dakotaensis) recently discovered in a soybean field in North Dakota, USA. Species-specific primers, targeting the internal transcribed spacer region of ribosomal DNA, were designed to be used in both conventional and quantitative real-time PCR assays for identification of P.dakotaensis. The specificity of the primers was evaluated in silico analysis and laboratory PCR experiments. Results showed that only P.dakotaensis DNA was exclusively amplified in conventional and real-time PCR assays but none of the DNA from other control species were amplified. Detection sensitivity analysis revealed that the conventional PCR was able to detect an equivalent to 1/8 of the DNA of a single nematode whereas real-time PCR detected an equivalent to 1/32 of the DNA of a single nematode. According to the generated standard curve the amplification efficiency of the primers in real-time PCR was 94% with a R2 value of 0.95 between quantification cycle number and log number of P.dakotaensis. To validate the assays to distinguish P.dakotaensis from other Pratylenchus spp. commonly detected in North Dakota soybean fields, 20 soil samples collected from seven counties were tested. The PCR assays amplified the DNA of P.dakotaensis and discriminated it from other Pratylenchus spp. present in North Dakota soybean fields. This is the first report of a species-specific and rapid PCR detection method suitable for use in diagnostic and research laboratories for the detection of P.dakotaensis.


Assuntos
DNA de Helmintos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Soja/parasitologia , Tylenchoidea/genética , Animais , Primers do DNA/síntese química , Primers do DNA/metabolismo , Limite de Detecção , North Dakota , Doenças das Plantas/parasitologia , Raízes de Plantas/parasitologia , Solo/parasitologia , Especificidade da Espécie , Tylenchoidea/classificação , Tylenchoidea/isolamento & purificação
20.
PLoS Biol ; 19(5): e3001236, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33961632

RESUMO

With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/genética , Primers do DNA , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Poliproteínas/genética , Proteínas Virais/genética
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