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1.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751106

RESUMO

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.


Assuntos
Betacoronavirus/genética , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/economia , Pneumonia Viral/diagnóstico , RNA Viral/análise , Betacoronavirus/isolamento & purificação , Colorimetria/economia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/metabolismo , Carga Viral
2.
Virus Res ; 288: 198129, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32822689

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.


Assuntos
Betacoronavirus/genética , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/métodos , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Técnicas de Laboratório Clínico/instrumentação , Colorimetria/instrumentação , Infecções por Coronavirus/virologia , Endodesoxirribonucleases/química , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Reologia , Sensibilidade e Especificidade
3.
Ecotoxicol Environ Saf ; 204: 111004, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32768745

RESUMO

Consumption of seafood contaminated with ciguatoxins (CTXs) leads to a foodborne disease known as ciguatera. Primary producers of CTXs are epibenthic dinoflagellates of the genera Gambierdiscus and Fukuyoa. In this study, thirteen Gambierdiscus and Fukuyoa strains were cultured, harvested at exponential phase, and CTXs were extracted with an implemented rapid protocol. Microalgal extracts were obtained from pellets with a low cell abundance (20,000 cell/mL) and were then analyzed with magnetic bead (MB)-based immunosensing tools (colorimetric immunoassay and electrochemical immunosensor). It is the first time that these approaches are used to screen Gambierdiscus and Fukuyoa strains, providing not only a global indication of the presence of CTXs, but also the ability to discriminate between two series of congeners (CTX1B and CTX3C). Analysis of the microalgal extracts revealed the presence of CTXs in 11 out of 13 strains and provided new information about Gambierdiscus and Fukuyoa toxin profiles. The use of immunosensing tools in the analysis of microalgal extracts facilitates the elucidation of further knowledge regarding these dinoflagellate genera and can contribute to improved ciguatera risk assessment and management.


Assuntos
Ciguatoxinas/isolamento & purificação , Colorimetria/métodos , Dinoflagelados/química , Técnicas Eletroquímicas/métodos , Imunoensaio/métodos , Ciguatoxinas/classificação , Especificidade da Espécie
4.
Virus Res ; 288: 198129, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: covidwho-719033

RESUMO

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.


Assuntos
Betacoronavirus/genética , Sistemas CRISPR-Cas , Técnicas de Laboratório Clínico/métodos , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Técnicas de Laboratório Clínico/instrumentação , Colorimetria/instrumentação , Infecções por Coronavirus/virologia , Endodesoxirribonucleases/química , Humanos , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , Reologia , Sensibilidade e Especificidade
5.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: covidwho-693630

RESUMO

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.


Assuntos
Betacoronavirus/genética , Colorimetria/métodos , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/economia , Pneumonia Viral/diagnóstico , RNA Viral/análise , Betacoronavirus/isolamento & purificação , Colorimetria/economia , Infecções por Coronavirus/virologia , Humanos , Limite de Detecção , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , Pneumonia Viral/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/metabolismo , Carga Viral
6.
Sci Transl Med ; 12(556)2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32719001

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Colorimetria/métodos , Colorimetria/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Proteínas do Nucleocapsídeo/genética , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , RNA-Seq , Sensibilidade e Especificidade , Pesquisa Médica Translacional
7.
Food Chem ; 332: 127392, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32623126

RESUMO

The present work describes a novel and rapid approach for evaluating total phenolic compounds (TPCs) in tea and fruits using colorimetric spots and the digital image-based (DIB) method. Colorimetric spots were formed by reacting diazotized aminobenzenes namely sulfanilic acid, sulfanilamide, or aniline with TPCs in the extract to form an azo dye. The limit of detection (LOD) was 6.5, 5.5, or 5.1 mg GAE (gallic acid equivalent) L-1 and the analytical range was 25-500, 20-500, or 18-200 mg GAE L-1, respectively. Correlation with the Folin-Ciocalteu assay was significant (Pearson coefficient, R = 0.970-0.991) while the antioxidant activity assay was moderate to high (R = 0.737-0.977). The method developed was successfully applied to the analysis of tea and fruits and showed RSD (n = 3) not exceeding 9.6, 8.5, and 9.7%, respectively. Ecologically, the DIB method developed could determine the variation of TPCs within cultivars and was found to be strongly dependent on the growing environment.


Assuntos
Benzeno/química , Colorimetria/métodos , Análise de Alimentos/métodos , Frutas/química , Fenóis/análise , Chá/química , Antioxidantes/análise , Limite de Detecção
8.
Food Chem ; 332: 127431, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32645668

RESUMO

Illegal usage of ß-agonists as the animal growth promoters can lead to multiple harmful impacts to public health, thus detection of ß-agonists at trace level in complex sample matrixes is of great importance. In recent years, emergence of advanced nanomaterials greatly facilitates the advancement of sensors in terms of sensitivity, specificity and robustness. Plenty of nanoparticles-based sensors have been developed for ß-agonists determination. In this review, we comprehensively summarized the construction of emerging nanoparticles-based sensors (including colorimetric sensors, fluorescent sensors, chemiluminescent sensors, electrochemical sensors, electrochemiluminescent sensors, surface enhanced Raman scattering sensors, surface plasmon resonance sensors, quartz crystal microbalance sensors, etc.), and nanomaterial-based enzyme-linked immunosorbent assay (nano-ELISA). Impressively, the applications of nanoparticles-based sensors and nano-ELISAs in the detection of ß-agonists have also been summarized and discussed. In the end, future opportunities and challenges in the design construction of nanoparticles (NPs)-based sensors and their applications in ß-agonist assay are tentatively proposed.


Assuntos
Agonistas Adrenérgicos/análise , Nanoestruturas/química , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Colorimetria/instrumentação , Colorimetria/métodos , Humanos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Análise Espectral Raman/instrumentação , Análise Espectral Raman/métodos , Ressonância de Plasmônio de Superfície/instrumentação , Ressonância de Plasmônio de Superfície/métodos
9.
PLoS One ; 15(7): e0235532, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32614905

RESUMO

The yeast Komagataella phaffii is widely used as a microbial host for heterologous protein production. However, molecular tools for this yeast are basically restricted to a few integrative and replicative plasmids. Four sequences that have recently been proposed as the K. phaffii centromeres could be used to develop a new class of mitotically stable vectors. In this work, we designed a color-based genetic assay to investigate plasmid stability in K. phaffii and constructed vectors bearing K. phaffii centromeres and the ADE3 marker. These genetic tools were evaluated in terms of mitotic stability by transforming an ade2/ade3 auxotrophic strain and regarding plasmid copy number by quantitative PCR (qPCR). Our results confirmed that the centromeric plasmids were maintained at low copy numbers as a result of typical chromosome-like segregation during cell division. These features, combined with in vivo assembly possibilities, prompt these plasmids as a new addition to the K. phaffii genetic toolbox.


Assuntos
Centrômero/genética , Colorimetria/métodos , Pichia/genética , Plasmídeos/análise , DNA Fúngico/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
10.
Sci Transl Med ; 12(556)2020 08 12.
Artigo em Inglês | MEDLINE | ID: covidwho-688785

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.


Assuntos
Betacoronavirus/genética , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Colorimetria/métodos , Colorimetria/estatística & dados numéricos , Infecções por Coronavirus/epidemiologia , Humanos , Técnicas de Diagnóstico Molecular/estatística & dados numéricos , Técnicas de Amplificação de Ácido Nucleico/estatística & dados numéricos , Proteínas do Nucleocapsídeo/genética , Pandemias , Pneumonia Viral/epidemiologia , RNA Viral/genética , RNA Viral/isolamento & purificação , RNA-Seq , Sensibilidade e Especificidade , Pesquisa Médica Translacional
11.
Food Chem ; 329: 127165, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504919

RESUMO

Biogenic amines are the important markers for food spoilage, thus, an on-package sensor for biogenic amine detection is crucial for food quality control. A dual detection platform including colorimetry and LDI-MS was developed for screening and quantitative determining of biogenic amines. Porous PLA film, was fabricated using calcium carbonate nanoparticles to enhance film porosity leading to increased surface area of colorimetric sensor. The color intensity significantly increases depending upon the enhanced analyte concentration with a linear range of 2.0-10.0 mg/mL for putrescine, and 0.1-6.0 mg/mL for cadaverine. On another layer, graphene oxide paper was applied as an LDI-MS substrate for sensitive quantification of biogenic amines. LOD values measured on graphene oxide coated side by LDI-MS were found to be 0.07 pM and 0.02 pM for putrescine and cadaverine, respectively. This platform was successfully applied for the detection of biogenic amines in pork samples with satisfactory results.


Assuntos
Aminas Biogênicas/análise , Colorimetria/métodos , Análise de Alimentos/métodos , Carne de Porco/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Cadaverina/análise , Qualidade dos Alimentos , Grafite , Limite de Detecção , Nanopartículas/química , Poliésteres/química , Porosidade , Putrescina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentação
12.
Food Chem ; 328: 127099, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32474238

RESUMO

In this study, we developed a competitive colorimetric immunoassay for qualitative detection of DAN based on oxidation of iron (Ⅱ) (Fe2+) in the presence of glucose oxidase (GOx) and color change induced by Fe2+-phenanthroline (Phen) chromogenic system. Streptavidin (SA) acted as a linker between biotinylated anti-DAN-monoantibody (bio-mAb) and biotinylated GOx (bio-GOx) to form the immunocomplexes bio-mAb-SA-bio-GOx. In the absence of DAN, the immunocomplexes bio-mAb-SA-bio-GOx combining with coated DAN-ovalbumin (DAN-OVA) will be immobilized and catalyze glucose to produce H2O2. Fe2+ is oxidized to Fe3+ by H2O2, giving rise to a colorless result. In the presence of DAN, Fe2+ produces a chelation reaction with Phen, leading to orange-red color. Under optimal conditions, the detection limit (LOD) by naked eyes was 2.5 ng mL-1 in milk, chicken, beef, and pork samples. Low LOD, no matrix effect, and no signal reader requirement make it possibly applied to quickly screen DAN on site.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , Fluoroquinolonas/análise , Glucose Oxidase/metabolismo , Imunoensaio/métodos , Quelantes de Ferro/química , Fenantrolinas/química , Biocatálise , Catálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fluoroquinolonas/química , Glucose/química , Glucose Oxidase/química , Peróxido de Hidrogênio/química , Limite de Detecção
13.
PLoS Negl Trop Dis ; 14(6): e0008364, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32492018

RESUMO

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera.


Assuntos
Colorimetria/métodos , Testes Diagnósticos de Rotina/métodos , Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/diagnóstico , Febre Hemorrágica da Crimeia/virologia , Replicação Viral , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vírus da Febre Hemorrágica da Crimeia-Congo/genética , Humanos , Imunoglobulina G/sangue , Modelos Moleculares , Nucleoproteínas/química , Nucleoproteínas/genética , Conformação Proteica
14.
Food Chem ; 331: 127090, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32593035

RESUMO

Chlorothalonil is a class of 2B carcinogen which is widely used in the prevention and treatment of fungal diseases in food samples. Its residual problem has been increasingly concerned by society. In this paper, a fast and simple colorimetric assay based on Manganese dioxide nanosheets (MnO2 NSs)-oxidize 3,3',5,5'-tetramethylbenzidine (TMB) platform was used to detect residual pesticide chlorothalonil in food samples. Under optimal conditions, the half maximal inhibitory concentration and the limit of detection of chlorothalonil were 3.27 and 0.024 ng/mL. There were no obvious cross-reactivity between chlorothalonil and interference substances. The recoveries shown the satisfactory results. The results of colorimetric assay for the authentic samples were largely consistent with gas chromatography. Therefore, the proposed method would be convenient and satisfactory analytical methods for the monitoring of chlorothalonil. Furthermore, the MnO2 - TMB system was used to produce test strips for quick and convenient visual detection of chlorothalonil with good performance.


Assuntos
Colorimetria/métodos , Análise de Alimentos/métodos , Compostos de Manganês/química , Nanoestruturas/química , Nitrilos/análise , Óxidos/química , Benzidinas/química , Colorimetria/instrumentação , Análise de Alimentos/instrumentação , Contaminação de Alimentos/análise , Fungicidas Industriais/análise , Limite de Detecção , Oxirredução , Oxirredutases/química
15.
Food Chem ; 328: 127149, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32480264

RESUMO

A novel approach for the discrimination of different glucosinolates (sinigrin, progoitrin, gluconapin, 4-methoxyglucobrassicin, glucoraphanin, glucobrassicin, glucoiberin, glucobrassicanapin, glucoraphenin, and glucoerucin) using a colorimetric sensor array (CSA) is reported herein. The developed CSA technique exhibited an acceptable linearity (r2 ≥ 0.97) over a concentration range of 0-150 µM for the 10 glucosinolates. The CSA coupled with principal component analysis and hierarchical cluster analysis correctly distinguished the majority of glucosinolate samples according to their type. In addition, the CSA coupled with linear discriminant analysis correctly classified the majority of 8 kinds of cruciferous vegetable samples with an overall accuracy of 94%. Furthermore, the partial least squares regression results showed that the CSA responses were correlated with the concentration in a correlation coefficient (Rp) range of 0.813-0.964. These results demonstrate that the described procedure based on the CSA technique could be useful for the rapid discrimination of different glucosinolates.


Assuntos
Brassicaceae/química , Colorimetria/métodos , Análise de Alimentos/métodos , Glucosinolatos/análise , Colorimetria/estatística & dados numéricos , Análise de Alimentos/estatística & dados numéricos , Análise dos Mínimos Quadrados , Análise de Componente Principal
16.
Food Chem ; 328: 126768, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470772

RESUMO

A colorimetric pH indicator was developed using nanofibers of poly(lactic acid) (PLA) and polyethylene oxide (PEO) combined with biomass of the microalga Spirulina sp. LEB 18. This study evaluates the potential use of microalgal biomass encapsulated in polymer nanofibers to develop a colorimetric pH indicator. Nanofibers containing the biomass were exposed to solutions with different pH values (pH 1-10), and color variations were measured using a colorimeter. The wettability analysis of the nanofibers showed hydrophilicity (zero angle with water), which allows ions to interact with the biomass, indicating a fast color response as a function of pH. When subjected to pH variations, indicators containing 1, 2 or 3% (w v-1) of biomass provided ΔΕ values >12, indicating an absolute difference in color. Therefore, this innovative material has the potential to be applied as a intelligent indicator to verify food quality through a visual signal of the product condition.


Assuntos
Colorimetria/métodos , Nanofibras/química , Spirulina/fisiologia , Biomassa , Cor , Concentração de Íons de Hidrogênio , Poliésteres/química , Polietilenoglicóis/química , Molhabilidade
17.
ACS Nano ; 14(6): 7617-7627, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32437124

RESUMO

The current outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) demands its rapid, convenient, and large-scale diagnosis to downregulate its spread within as well as across the communities. But the reliability, reproducibility, and selectivity of majority of such diagnostic tests fail when they are tested either to a viral load at its early representation or to a viral gene mutated during its current spread. In this regard, a selective "naked-eye" detection of SARS-CoV-2 is highly desirable, which can be tested without accessing any advanced instrumental techniques. We herein report the development of a colorimetric assay based on gold nanoparticles (AuNPs), when capped with suitably designed thiol-modified antisense oligonucleotides (ASOs) specific for N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2, could be used for diagnosing positive COVID-19 cases within 10 min from the isolated RNA samples. The thiol-modified ASO-capped AuNPs agglomerate selectively in the presence of its target RNA sequence of SARS-CoV-2 and demonstrate a change in its surface plasmon resonance. Further, the addition of RNaseH cleaves the RNA strand from the RNA-DNA hybrid leading to a visually detectable precipitate from the solution mediated by the additional agglomeration among the AuNPs. The selectivity of the assay has been monitored in the presence of MERS-CoV viral RNA with a limit of detection of 0.18 ng/µL of RNA having SARS-CoV-2 viral load. Thus, the current study reports a selective and visual "naked-eye" detection of COVID-19 causative virus, SARS-CoV-2, without the requirement of any sophisticated instrumental techniques.


Assuntos
Betacoronavirus/genética , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/diagnóstico , Nanopartículas Metálicas , Proteínas do Nucleocapsídeo/genética , Oligonucleotídeos Antissenso/genética , Pneumonia Viral/diagnóstico , Sequência de Bases , Betacoronavirus/isolamento & purificação , Colorimetria/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Genes Virais , Ouro , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Nanotecnologia/métodos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Capuzes de RNA/genética , RNA Viral/genética , Ressonância de Plasmônio de Superfície/métodos
18.
ACS Nano ; 14(6): 7617-7627, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: covidwho-647565

RESUMO

The current outbreak of the pandemic coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) demands its rapid, convenient, and large-scale diagnosis to downregulate its spread within as well as across the communities. But the reliability, reproducibility, and selectivity of majority of such diagnostic tests fail when they are tested either to a viral load at its early representation or to a viral gene mutated during its current spread. In this regard, a selective "naked-eye" detection of SARS-CoV-2 is highly desirable, which can be tested without accessing any advanced instrumental techniques. We herein report the development of a colorimetric assay based on gold nanoparticles (AuNPs), when capped with suitably designed thiol-modified antisense oligonucleotides (ASOs) specific for N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2, could be used for diagnosing positive COVID-19 cases within 10 min from the isolated RNA samples. The thiol-modified ASO-capped AuNPs agglomerate selectively in the presence of its target RNA sequence of SARS-CoV-2 and demonstrate a change in its surface plasmon resonance. Further, the addition of RNaseH cleaves the RNA strand from the RNA-DNA hybrid leading to a visually detectable precipitate from the solution mediated by the additional agglomeration among the AuNPs. The selectivity of the assay has been monitored in the presence of MERS-CoV viral RNA with a limit of detection of 0.18 ng/µL of RNA having SARS-CoV-2 viral load. Thus, the current study reports a selective and visual "naked-eye" detection of COVID-19 causative virus, SARS-CoV-2, without the requirement of any sophisticated instrumental techniques.


Assuntos
Betacoronavirus/genética , Técnicas Biossensoriais/métodos , Infecções por Coronavirus/diagnóstico , Nanopartículas Metálicas , Proteínas do Nucleocapsídeo/genética , Oligonucleotídeos Antissenso/genética , Pneumonia Viral/diagnóstico , Sequência de Bases , Betacoronavirus/isolamento & purificação , Colorimetria/métodos , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Genes Virais , Ouro , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Nanotecnologia/métodos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Capuzes de RNA/genética , RNA Viral/genética , Ressonância de Plasmônio de Superfície/métodos
19.
J Mol Diagn ; 22(6): 729-735, 2020 06.
Artigo em Inglês | MEDLINE | ID: covidwho-214148

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.


Assuntos
Betacoronavirus/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Colorimetria/métodos , Primers do DNA , Violeta Genciana , RNA Viral/genética , Transcrição Reversa
20.
Med. clín (Ed. impr.) ; 154(7): 275-278, abr. 2020. graf
Artigo em Espanhol | IBECS | ID: ibc-190912

RESUMO

INTRODUCCIÓN Y OBJETIVOS: La analbuminemia congénita (AAC) (MIM #616000) es una enfermedad autosómica recesiva (prevalencia <1/106) causada por defectos en el gen ALB que implican la ausencia o marcada disminución de la albuminemia. En este artículo, describimos un caso de AAC detectado en nuestro hospital. MATERIAL Y MÉTODOS: Mujer de 42 años con hipoproteinemia e hipoalbuminemia de causa no filiada. El estudio bioquímico se realizó siguiendo las técnicas y los controles de calidad habituales de nuestro laboratorio: albuminemia (colorimetría y nefelometría); electroforesis de proteínas (capilar y gel de agarosa) y análisis molecular del gen ALB (extracción de ADN y amplificación PCR de los 14 exones codificantes más regiones intrónicas adyacentes y secuenciación Sanger). RESULTADOS: Descartadas las causas más frecuentes de hipoalbuminemia, se confirmó la analbuminemia por electroforesis y nefelometría. El estudio molecular del gen ALB evidenció la presencia de la variante c.1289+1G>A (variante Guimarães) en homozigosis. CONCLUSIONES: Este es el primer caso confirmado mediante estudio molecular de AAC en España. La paciente presenta la variante Guimarães descrita previamente en otros 4 pacientes en el mundo


INTRODUCTION AND OBJECTIVES: Congenital analbuminaemia (CCA) (MIM #616000) is an autosomal recessive disorder (prevalence < 1/106) caused by defects in the ALB gene leading to absence or severe reduction of albuminaemia. This paper describes a case of CCA detected and diagnosed in our hospital. MATERIALS AND METHODS: A 42-year old woman showing hypoproteinaemia and hypoalbuminaemia of unknown aetiology. Biochemical study was performed according to routine quality controlled analytical procedures: Albuminaemia (colorimetric and nephelometric methods). Protein electrophoresis (capillary and agarose gel). Molecular study of the ALB gene: DNA extraction, PCR amplification of the 14 coding exons plus adjacent intron regions and Sanger sequencing. RESULTS: After discarding the most common causes of hypoalbuminaemia, the analbuminaemia was confirmed by nephelometry and protein electrophoresis. The proband was found to be homozygous for molecular defect in the ALB gene: variant c.1289+1G>A previously reported as Guimarães variant. CONCLUSIONS: This is the first case of CCA confirmed by molecular study in Spain. The proband shows the Guimarães variant previously described in 4 patients worldwide


Assuntos
Humanos , Feminino , Adulto , Doenças Genéticas Inatas/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Albumina Sérica/genética , Doenças Genéticas Inatas/genética , Eletroforese , Hipoalbuminemia/etiologia , Colorimetria/métodos , Nefelometria e Turbidimetria/métodos , Diagnóstico Diferencial
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