Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 112
Filtrar
Añadir filtros

Base de datos
Intervalo de año
1.
Front Cell Infect Microbiol ; 11: 613304, 2021.
Artículo en Inglés | MEDLINE | ID: covidwho-1088903

RESUMEN

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


Asunto(s)
/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /genética , /virología , /genética , Cartilla de ADN/genética , Humanos , Fosfoproteínas/genética , Pruebas en el Punto de Atención , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/instrumentación , Recombinasas/metabolismo , Transcripción Reversa , Sensibilidad y Especificidad
2.
Sci Adv ; 7(7)2021 02.
Artículo en Inglés | MEDLINE | ID: covidwho-1084597

RESUMEN

We present INSIGHT [isothermal NASBA (nucleic acid sequence-based amplification) sequencing-based high-throughput test], a two-stage coronavirus disease 2019 testing strategy, using a barcoded isothermal NASBA reaction. It combines point-of-care diagnosis with next-generation sequencing, aiming to achieve population-scale testing. Stage 1 allows a quick decentralized readout for early isolation of presymptomatic or asymptomatic patients. It gives results within 1 to 2 hours, using either fluorescence detection or a lateral flow readout, while simultaneously incorporating sample-specific barcodes. The same reaction products from potentially hundreds of thousands of samples can then be pooled and used in a highly multiplexed sequencing-based assay in stage 2. This second stage confirms the near-patient testing results and facilitates centralized data collection. The 95% limit of detection is <50 copies of viral RNA per reaction. INSIGHT is suitable for further development into a rapid home-based, point-of-care assay and is potentially scalable to the population level.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Pruebas en el Punto de Atención , /genética , /diagnóstico , Humanos
3.
Int J Nanomedicine ; 16: 383-402, 2021.
Artículo en Inglés | MEDLINE | ID: covidwho-1076350

RESUMEN

Advancements in analytical diagnostic systems for point-of-care (POC) application have gained considerable attention because of their rapid operation at the site required to manage severe diseases, even in a personalized manner. The POC diagnostic devices offer easy operation, fast analytical outcome, and affordable cost, which promote their advanced research and versatile adoptability. Keeping advantages in view, considerable efforts are being made to design and develop smart sensing components such as miniaturized transduction, interdigitated electrodes-based sensing chips, selective detection at low level, portable packaging, and sustainable durability to promote POC diagnostics according to the needs of patient care. Such effective diagnostics systems are in demand, which creates the challenge to make them more efficient in every aspect to generate a desired bio-informatic needed for better health access and management. Keeping advantages and scope in view, this mini review focuses on practical scenarios associated with miniaturized analytical diagnostic devices at POC application for targeted disease diagnostics smartly and efficiently. Moreover, advancements in technologies, such as smartphone-based operation, paper-based sensing assays, and lab-on-a-chip (LOC) which made POC more sensitive, informative, and suitable for major infectious disease diagnosis, are the main focus here. Besides, POC diagnostics based on automated patient sample integration with a sensing platform is continuously improving therapeutics interventions against specific infectious disease. This review also discussed challenges associated with state-of-the-art technology along with future research opportunities to design and develop next generation POC diagnostic systems needed to manage infectious diseases in a personalized manner.


Asunto(s)
Pruebas en el Punto de Atención , Medicina de Precisión/métodos , Enfermedades Transmisibles/diagnóstico , Humanos , Dispositivos Laboratorio en un Chip , Teléfono Inteligente
5.
Epidemiol Prev ; 44(5-6 Suppl 2): 193-199, 2020.
Artículo en Inglés | MEDLINE | ID: covidwho-1068139

RESUMEN

BACKGROUND: facing the SARS-CoV-2 epidemic requires intensive testing on the population to early identify and isolate infected subjects. Although RT-PCR is the most reliable technique to detect ongoing infections, serological tests are frequently proposed as tools in heterogeneous screening strategies. OBJECTIVES: to analyse the performance of a screening strategy proposed by the local government of Tuscany (Central Italy), which first uses qualitative rapid tests for antibody detection, and then RT-PCR tests on the positive subjects. METHODS: a simulation study is conducted to investigate the number of RT-PCR tests required by the screening strategy and the undetected ongoing infections in a pseudo-population of 500,000 subjects, under different prevalence scenarios and assuming a sensitivity of the serological test ranging from 0.50 to 0.80 (specificity 0.98). A compartmental model is used to predict the number of new infections generated by the false negatives two months after the screening, under different values of the infection reproduction number. RESULTS: assuming a sensitivity equal to 0.80 and a prevalence of 0.3%, the screening procedure would require on average 11,167 RT-PCR tests and would produce 300 false negatives, responsible after two months of a number of contagions ranging from 526 to 1,132, under the optimistic scenario of a reproduction number between 0.5 to 1. Resources and false negatives increase with the prevalence. CONCLUSIONS: the analysed screening procedure should be avoided unless the prevalence and the rate of contagion are very low. The cost and effectiveness of the screening strategies should be evaluated in the actual context of the epidemic, accounting for the fact that it may change over time.


Asunto(s)
Anticuerpos Antivirales/sangre , Simulación por Computador , Tamizaje Masivo/métodos , Modelos Teóricos , Pandemias , /inmunología , Número Básico de Reproducción , /transmisión , /métodos , Análisis Costo-Beneficio , Reacciones Falso Negativas , Reacciones Falso Positivas , Humanos , Italia/epidemiología , Tamizaje Masivo/economía , Método de Montecarlo , Pruebas en el Punto de Atención/economía , Prevalencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad
6.
Anal Chim Acta ; 1146: 184-199, 2021 Feb 15.
Artículo en Inglés | MEDLINE | ID: covidwho-1064681

RESUMEN

The COVID-19 global pandemic of 2019-2020 pointedly revealed the lack of diagnostic solutions that are able to keep pace with the rapid spread of the virus. Despite the promise of decades of lab-on-a-chip research, no commercial products were available to deliver rapid results or enable testing in the field at the onset of the pandemic. In this critical review, we assess the current state of progress on the development of point-of-care technologies for the diagnosis of viral diseases that cause pandemics. While many previous reviews have reported on progress in various lab-on-a-chip technologies, here we address the literature from the perspective of the testing needs of a rapidly expanding pandemic. First, we recommend a set of requirements to heed when designing point-of-care diagnostic technologies to address the testing needs of a pandemic. We then review the current state of assay technologies with a focus on isothermal amplification and lateral-flow immunoassays. Though there is much progress on assay development, we argue that the largest roadblock to deployment exists in sample preparation. We summarize current approaches to automate sample preparation and discuss both the progress and shortcomings of these developments. Finally, we provide our recommendations to the field of specific challenges to address in order to prepare for the next pandemic.


Asunto(s)
/diagnóstico , Pandemias , Sistemas de Atención de Punto/tendencias , Pruebas en el Punto de Atención/tendencias , Humanos , Dispositivos Laboratorio en un Chip
8.
Sci Adv ; 7(2)2021 01.
Artículo en Inglés | MEDLINE | ID: covidwho-1066788

RESUMEN

Point-of-care COVID-19 assays that are more sensitive than the current RT-PCR (reverse transcription polymerase chain reaction) gold standard assay are needed to improve disease control efforts. We describe the development of a portable, ultrasensitive saliva-based COVID-19 assay with a 15-min sample-to-answer time that does not require RNA isolation or laboratory equipment. This assay uses CRISPR-Cas12a activity to enhance viral amplicon signal, which is stimulated by the laser diode of a smartphone-based fluorescence microscope device. This device robustly quantified viral load over a broad linear range (1 to 105 copies/µl) and exhibited a limit of detection (0.38 copies/µl) below that of the RT-PCR reference assay. CRISPR-read SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) RNA levels were similar in patient saliva and nasal swabs, and viral loads measured by RT-PCR and the smartphone-read CRISPR assay demonstrated good correlation, supporting the potential use of this portable assay for saliva-based point-of-care COVID-19 diagnosis.


Asunto(s)
/diagnóstico , Pruebas en el Punto de Atención , Saliva/virología , Teléfono Inteligente , Animales , Sistemas CRISPR-Cas , Chlorocebus aethiops , Simulación por Computador , Femenino , Humanos , Límite de Detección , Macaca mulatta , Masculino , Técnicas de Diagnóstico Molecular/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Células Vero , Carga Viral
9.
J Am Chem Soc ; 143(4): 1722-1727, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: covidwho-1065802

RESUMEN

The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.


Asunto(s)
Técnicas Biosensibles/métodos , /virología , Técnicas Electroquímicas/métodos , Virión/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Técnicas Electroquímicas/instrumentación , Electrodos , Humanos , Pruebas en el Punto de Atención , Saliva/virología
10.
J Infect Dis ; 223(2): 206-213, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: covidwho-1060913

RESUMEN

BACKGROUND: Recent advances in CRISPR-based diagnostics suggest that DETECTR, a combination of reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) and subsequent Cas12 bystander nuclease activation by amplicon-targeting ribonucleoprotein complexes, could be a faster and cheaper alternative to quantitative reverse-transcription polymerase chain reaction (qRT-PCR) without sacrificing sensitivity and/or specificity. METHODS: In this study, we compare DETECTR with qRT-PCR to diagnose coronavirus disease 2019 on 378 patient samples. Patient sample dilution assays suggest a higher analytical sensitivity of DETECTR compared with qRT-PCR; however, this was not confirmed in this large patient cohort, where we report 95% reproducibility between the 2 tests. RESULTS: These data showed that both techniques are equally sensitive in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) providing additional value of DETECTR to the currently used qRT-PCR platforms. For DETECTR, different guide ribonucleic acids can be used simultaneously to obviate negative results due to mutations in N-gene. Lateral flow strips, suitable as a point-of-care test, showed a 100% correlation to the high-throughput DETECTR assay. More importantly, DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses. CONCLUSIONS: Because there is no need for specialized equipment, DETECTR could be rapidly implemented as a complementary technically independent approach to qRT-PCR thereby increasing the testing capacity of medical microbiological laboratories and relieving the existent PCR platforms for routine non-SARS-CoV-2 diagnostic testing.


Asunto(s)
/métodos , /virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Pruebas en el Punto de Atención , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Estándares de Referencia , Reproducibilidad de los Resultados , /genética
11.
Nat Commun ; 12(1): 724, 2021 02 01.
Artículo en Inglés | MEDLINE | ID: covidwho-1060236

RESUMEN

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.


Asunto(s)
Técnicas Biosensibles/métodos , Redes Reguladoras de Genes/genética , Glucosa/análisis , Ácidos Nucleicos/análisis , Sistemas de Atención de Punto , Pruebas en el Punto de Atención , Técnicas Biosensibles/instrumentación , /epidemiología , Glucosa/metabolismo , Humanos , Ácidos Nucleicos/genética , Pandemias , /fisiología , Fiebre Tifoidea/sangre , Fiebre Tifoidea/diagnóstico , Fiebre Tifoidea/microbiología
12.
Sci Rep ; 11(1): 2402, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: covidwho-1054048

RESUMEN

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


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/aislamiento & purificación , /genética , Técnicas de Laboratorio Clínico/métodos , Técnicas y Procedimientos Diagnósticos , Pruebas Diagnósticas de Rutina , Humanos , Límite de Detección , Pruebas en el Punto de Atención , ARN Viral/genética , Transcripción Reversa , /metabolismo , Sensibilidad y Especificidad
14.
Eur Rev Med Pharmacol Sci ; 25(1): 503-517, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: covidwho-1052577

RESUMEN

OBJECTIVE: To evaluate the diagnostic accuracy of the Food and Drug Administration Emergency Use Authorization (FDA-EUA) authorized point-of-care tests (POCTs) for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MATERIALS AND METHODS: A systematic literature search was conducted using the PubMed, Embase, and Web of Science databases for articles published till August 10, 2020. We included studies providing information regarding diagnostic test accuracy of FDA-EUA POCTs for SARS-CoV-2 detection. The methodologic quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 tool. The review protocol is registered in the International Prospective Register of Systematic Reviews (protocol number CRD42020202248). RESULTS: We included 26 studies describing a total of 3242 samples. The summary sensitivity and specificity were 0.94 [95% confidence interval (CI): 0.88-0.97] and 1.00 (95% CI: 0.99-1.00), respectively. The area under the summary receiver operating characteristic curve was 1.00 (95% CI: 0.99-1.00). A pooled analysis based on the index test revealed a summary sensitivity and specificity of Cepheid Xpert Xpress SARS-CoV-2 [0.99 (95% CI: 0.97-1.00) and 0.99 (95% CI: 0.94-1.00, respectively)] and ID NOW COVID-19 [0.78 (95% CI: 0.74-0.82) and 1.00 (95% CI: 0.98-1.00), respectively]. CONCLUSIONS: FDA-EUA POCTs, especially molecular assays, have high sensitivity, specificity, and overall diagnostic accuracy for detecting SARS-CoV-2. If approved, FDA-EUA POCTs can provide a rapid and practical way to identify infected individuals early on and help to limit the strain on the healthcare system. However, more high-quality clinical data are required to support our results.


Asunto(s)
/métodos , /diagnóstico , Pruebas en el Punto de Atención/normas , /aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Técnicas de Laboratorio Clínico/normas , Humanos , Garantía de la Calidad de Atención de Salud , Sensibilidad y Especificidad , Estados Unidos , United States Food and Drug Administration
15.
PLoS One ; 16(1): e0245848, 2021.
Artículo en Inglés | MEDLINE | ID: covidwho-1052440

RESUMEN

BACKGROUND: COVID-19 (COronaVIrus Disease 2019) is an infectious respiratory disease caused by the novel SARS-CoV-2 virus. Point of Care (POC) tests have been developed to detect specific antibodies, IgG and IgM, to SARS-CoV-2 virus in human whole blood. They need to be easily usable by the general population in order to alleviate the lockdown that many countries have initiated in response to the growing COVID-19 pandemic. A real-life study has been conducted in order to evaluate the performance of the COVID-PRESTO® POC test and the results were recently published. Even if this test showed very high sensitivity and specificity in a laboratory setting when used by trained professionals, it needs to be further evaluated for practicability when used by the general public in order to be approved by health authorities for in-home use. METHODS: 143 participants were recruited between March 2020 and April 2020 among non-medical populations in central France (nuclear plant workers, individuals attending the Orleans University Hospital vaccination clinic and Orleans University Hospital non-medical staff). Instructions for use, with or without a tutorial video, were made available to the volunteers. Two separate objectives were pursued: evaluation of the capability of participants to obtain an interpretable result, and evaluation of the users' ability to read the results. RESULTS: 88.4% of the test users judged the instructions for use leaflet to be clear and understandable. 99.3% of the users obtained a valid result and, according to the supervisors, 92.7% of the tests were properly performed by the users. Overall, 95% of the users gave positive feedback on the COVID PRESTO® as a potential self-test. Neither age nor education had an influence. CONCLUSION: COVID-PRESTO® was successfully used by an overwhelming majority of participants and its use was judged very satisfactory, therefore showing promising potential as a self-test to be used by the general population. This POC test can become an easy-to-use tool to help detect whether individuals are protected or not, particularly in the context of a second wave or a mass vaccination program.


Asunto(s)
/diagnóstico , /aislamiento & purificación , Adulto , Anciano , /epidemiología , Control de Enfermedades Transmisibles , Femenino , Francia/epidemiología , Pruebas Hematológicas/métodos , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Masculino , Persona de Mediana Edad , Pandemias/prevención & control , Pruebas en el Punto de Atención , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
17.
Anal Chem ; 93(5): 2950-2958, 2021 02 09.
Artículo en Inglés | MEDLINE | ID: covidwho-1041144

RESUMEN

There is an urgent need for ultrarapid testing regimens to detect the severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] infections in real-time within seconds to stop its spread. Current testing approaches for this RNA virus focus primarily on diagnosis by RT-qPCR, which is time-consuming, costly, often inaccurate, and impractical for general population rollout due to the need for laboratory processing. The latency until the test result arrives with the patient has led to further virus spread. Furthermore, latest antigen rapid tests still require 15-30 min processing time and are challenging to handle. Despite increased polymerase chain reaction (PCR)-test and antigen-test efforts, the pandemic continues to evolve worldwide. Herein, we developed a superfast, reagent-free, and nondestructive approach of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy with subsequent chemometric analysis toward the prescreening of virus-infected samples. Contrived saliva samples spiked with inactivated γ-irradiated COVID-19 virus particles at levels down to 1582 copies/mL generated infrared (IR) spectra with a good signal-to-noise ratio. Predominant virus spectral peaks are tentatively associated with nucleic acid bands, including RNA. At low copy numbers, the presence of a virus particle was found to be capable of modifying the IR spectral signature of saliva, again with discriminating wavenumbers primarily associated with RNA. Discrimination was also achievable following ATR-FTIR spectral analysis of swabs immersed in saliva variously spiked with virus. Next, we nested our test system in a clinical setting wherein participants were recruited to provide demographic details, symptoms, parallel RT-qPCR testing, and the acquisition of pharyngeal swabs for ATR-FTIR spectral analysis. Initial categorization of swab samples into negative versus positive COVID-19 infection was based on symptoms and PCR results (n = 111 negatives and 70 positives). Following training and validation (using n = 61 negatives and 20 positives) of a genetic algorithm-linear discriminant analysis (GA-LDA) algorithm, a blind sensitivity of 95% and specificity of 89% was achieved. This prompt approach generates results within 2 min and is applicable in areas with increased people traffic that require sudden test results such as airports, events, or gate controls.


Asunto(s)
Algoritmos , /fisiología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Virión/química , /virología , Análisis Discriminante , Rayos gamma , Humanos , Pruebas en el Punto de Atención , Análisis de Componente Principal , Saliva/virología , Sensibilidad y Especificidad , Relación Señal-Ruido , Virión/efectos de la radiación , Inactivación de Virus
18.
Biosens Bioelectron ; 177: 113005, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: covidwho-1033431

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic has been a major public health challenge in 2020. Early diagnosis of COVID-19 is the most effective method to control disease spread and prevent further mortality. As such, a high-precision and rapid yet economic assay method is urgently required. Herein, we propose an innovative method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using isothermal amplification of nucleic acids on a mesh containing multiple microfluidic pores. Hybridization of pathogen DNA and immobilized probes forms a DNA hydrogel by rolling circle amplification and, consequently, blocks the pores to prevent fluid movement, as observed. Following optimization of several factors, including pore size, mesh location, and precision microfluidics, the limit of detection (LOD) for SARS-CoV-2 was determined to be 0.7 aM at 15-min incubation. These results indicate rapid, easy, and effective detection with a moderate-sized LOD of the target pathogen by remote point-of-care testing and without the requirement of any sophisticated device.


Asunto(s)
/métodos , Hidrogeles/química , Ácidos Nucleicos Inmovilizados/química , Pruebas en el Punto de Atención , /aislamiento & purificación , Técnicas Biosensibles/economía , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , /economía , Sondas de ADN/química , Sondas de ADN/genética , Diseño de Equipo , Humanos , Ácidos Nucleicos Inmovilizados/genética , Dispositivos Laboratorio en un Chip , Límite de Detección , Técnicas de Diagnóstico Molecular/economía , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/economía , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/métodos , /genética
19.
PLoS One ; 16(1): e0243712, 2021.
Artículo en Inglés | MEDLINE | ID: covidwho-1024413

RESUMEN

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT-PCR (Reverse transcription-Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT-LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross-reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called "COVIDISC" to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.


Asunto(s)
/instrumentación , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Pruebas en el Punto de Atención , ARN Viral/genética , /genética , /economía , Diseño de Equipo , Humanos , Límite de Detección , Técnicas de Diagnóstico Molecular/economía , Técnicas de Amplificación de Ácido Nucleico/economía , Pruebas en el Punto de Atención/economía , ARN Viral/aislamiento & purificación , Factores de Tiempo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA