Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.325
Filtrar
1.
PLoS One ; 16(3): e0248042, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33657176

RESUMEN

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


Asunto(s)
/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /genética , /genética , Técnicas de Laboratorio Clínico/métodos , Infecciones por Coronavirus/virología , Cartilla de ADN/genética , Humanos , Proteínas de la Nucleocápside/genética , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transcripción Reversa/genética , Sensibilidad y Especificidad
2.
Nat Commun ; 12(1): 1467, 2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33674580

RESUMEN

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.


Asunto(s)
/métodos , /virología , Colorimetría/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico/métodos , Pruebas en el Punto de Atención , /aislamiento & purificación , /genética , Humanos , Fosfoproteínas/genética , ARN Viral/genética , Sensibilidad y Especificidad
3.
Nat Commun ; 12(1): 1739, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741959

RESUMEN

Extensive testing is essential to break the transmission of SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Here, we present a CRISPR-based diagnostic assay that is robust to viral genome mutations and temperature, produces results fast, can be applied directly on nasopharyngeal (NP) specimens without RNA purification, and incorporates a human internal control within the same reaction. Specifically, we show that the use of an engineered AsCas12a enzyme enables detection of wildtype and mutated SARS-CoV-2 and allows us to perform the detection step with loop-mediated isothermal amplification (LAMP) at 60-65 °C. We also find that the use of hybrid DNA-RNA guides increases the rate of reaction, enabling our test to be completed within 30 minutes. Utilizing clinical samples from 72 patients with COVID-19 infection and 57 healthy individuals, we demonstrate that our test exhibits a specificity and positive predictive value of 100% with a sensitivity of 50 and 1000 copies per reaction (or 2 and 40 copies per microliter) for purified RNA samples and unpurified NP specimens respectively.


Asunto(s)
/métodos , ARN Guia , /genética , Proteínas Bacterianas/genética , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Endodesoxirribonucleasas/genética , Humanos , Técnicas de Diagnóstico Molecular/métodos , Mutación , Nasofaringe/virología , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , Sensibilidad y Especificidad
4.
Biotechniques ; 70(3): 167-174, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33535813

RESUMEN

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


Asunto(s)
Virus de la Influenza A/genética , Virus de la Influenza B/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , /genética , /métodos , Cartilla de ADN/genética , Fluorescencia , ARN Viral/análisis , Transcripción Reversa , Sensibilidad y Especificidad
5.
Biosens Bioelectron ; 179: 113099, 2021 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-33640656

RESUMEN

The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/µL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/µL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.


Asunto(s)
/instrumentación , Dispersión Dinámica de Luz/instrumentación , Emulsiones/química , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , /aislamiento & purificación , Técnicas Biosensibles/economía , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , /métodos , Dispersión Dinámica de Luz/economía , Dispersión Dinámica de Luz/métodos , Diseño de Equipo , Humanos , Límite de Detección , Técnicas de Diagnóstico Molecular/economía , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/economía , Técnicas de Amplificación de Ácido Nucleico/métodos , Teléfono Inteligente , Factores de Tiempo
6.
Viruses ; 13(2)2021 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-33562285

RESUMEN

Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions.


Asunto(s)
Genoma Viral/genética , Metagenómica/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Virus/aislamiento & purificación , Biblioteca Genómica , Humanos , Límite de Detección , Virus/genética
8.
J Clin Virol ; 136: 104760, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33610926

RESUMEN

The new coronavirus infection (COVID-19) is a major public health concern, with a high burden and risk for infection among patients and healthcare workers. Saliva droplets containing SARS-COV-2 are a major vector for COVID-19 infection, making saliva a promising alternative for COVID-19 testing using nasopharyngeal swab samples. To diagnose COVID-19 patients in the field, a point-of-care test (POCT) using saliva was conceptualized. We have developed a simple method for extracting RNA from saliva samples using semi-alkaline proteinase, a sputum homogenizer typically used for preparing samples for tuberculosis testing, and a subsequent simple heating step with no need for centrifugation or RNA extraction. Further, we newly developed a triplex reverse transcription loop-mediated isothermal amplification approach (RT-LAMP) which utilizes colorimetric readout using a heat block, with results evaluated with the unaided eye. In 44 clinical patients suspected of having COVID-19 infection, the test took 45 min, and resulted in a diagnostic sensitivity of 82.6% (19/23) and diagnostic specificity of 100% (21/21), compared to the reference standard. The limit of detection was 250 copies/reaction (25,000 copies/mL). Our newly developed POCT approach achieved simple RNA extraction and constant RT-LAMP detection. This POCT has the potential to be used for simple inspection stations in a field setting, helping reduce the risk of infection by simplifying and accelerating testing for COVID-19.


Asunto(s)
/métodos , Pruebas en el Punto de Atención , ARN Viral/análisis , Saliva/virología , Humanos , Límite de Detección , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /aislamiento & purificación
9.
Talanta ; 225: 121978, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33592726

RESUMEN

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.


Asunto(s)
Infecciones por Caliciviridae/diagnóstico , Colorimetría/métodos , Gastroenteritis/diagnóstico , Infecciones por Caliciviridae/virología , Colorimetría/economía , Colorimetría/instrumentación , Análisis Costo-Beneficio , Gastroenteritis/virología , Genotipo , Humanos , Norovirus/genética , Norovirus/fisiología , Técnicas de Amplificación de Ácido Nucleico/métodos , Papel , ARN Viral/genética , Sensibilidad y Especificidad
10.
Anal Chem ; 93(8): 4126-4133, 2021 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-33570401

RESUMEN

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


Asunto(s)
Sistemas CRISPR-Cas , Colorimetría/métodos , ADN/química , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/análisis , /aislamiento & purificación , Proteínas Bacterianas , Secuencia de Bases , Proteínas Asociadas a CRISPR , ADN/genética , Endodesoxirribonucleasas , Oro/química , Humanos , Nanopartículas del Metal/química , Fosfoproteínas/genética , Poliproteínas/genética , ARN Viral/genética , Transcripción Reversa , Resonancia por Plasmón de Superficie , Proteínas Virales/genética
11.
Biosens Bioelectron ; 178: 113049, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33540323

RESUMEN

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.


Asunto(s)
Técnicas Biosensibles/métodos , /diagnóstico , Polarización de Fluorescencia/métodos , /aislamiento & purificación , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/estadística & datos numéricos , /instrumentación , Sistemas CRISPR-Cas , Diseño de Equipo , Polarización de Fluorescencia/instrumentación , Polarización de Fluorescencia/estadística & datos numéricos , Humanos , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/estadística & datos numéricos , Pandemias , Sistemas de Atención de Punto/estadística & datos numéricos , Procesamiento de Señales Asistido por Computador , Relación Señal-Ruido
12.
Biosens Bioelectron ; 178: 113041, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33545551

RESUMEN

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.


Asunto(s)
/métodos , /virología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /aislamiento & purificación , Técnicas Biosensibles/métodos , Técnicas Biosensibles/estadística & datos numéricos , Humanos , Límite de Detección , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena de la Polimerasa Multiplex/estadística & datos numéricos , Técnicas de Amplificación de Ácido Nucleico/estadística & datos numéricos , Pandemias , Sistemas de Atención de Punto/estadística & datos numéricos , ARN Viral/análisis , ARN Viral/genética , Sensibilidad y Especificidad
13.
PLoS One ; 16(2): e0247792, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33635936

RESUMEN

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.


Asunto(s)
/métodos , Endopeptidasa K/química , /aislamiento & purificación , Animales , Chlorocebus aethiops , Calor , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Sensibilidad y Especificidad , Manejo de Especímenes/métodos , Células Vero
14.
Sci Rep ; 11(1): 2936, 2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33536475

RESUMEN

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.


Asunto(s)
/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/metabolismo , /genética , /virología , Humanos , Límite de Detección , /aislamiento & purificación
15.
Nat Commun ; 12(1): 802, 2021 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-33547323

RESUMEN

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/µL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.


Asunto(s)
Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , /aislamiento & purificación , /virología , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , /fisiología , Sensibilidad y Especificidad
16.
J Vis Exp ; (168)2021 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-33616108

RESUMEN

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


Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Ácidos Nucleicos/genética , Impresión Tridimensional , Transcripción Reversa/genética , /aislamiento & purificación , /genética , Recursos en Salud , Humanos , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos
17.
PLoS One ; 16(2): e0240524, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33626039

RESUMEN

Managing the pandemic caused by SARS-CoV-2 requires new capabilities in testing, including the possibility of identifying, in minutes, infected individuals as they enter spaces where they must congregate in a functioning society, including workspaces, schools, points of entry, and commercial business establishments. Here, the only useful tests (a) require no sample transport, (b) require minimal sample manipulation, (c) can be performed by unlicensed individuals, (d) return results on the spot in much less than one hour, and (e) cost no more than a few dollars. The sensitivity need not be as high as normally required by the FDA for screening asymptomatic carriers (as few as 10 virions per sample), as these viral loads are almost certainly not high enough for an individual to present a risk for forward infection. This allows tests specifically useful for this pandemic to trade-off unneeded sensitivity for necessary speed, simplicity, and frugality. In some studies, it was shown that viral load that creates forward-infection risk may exceed 105 virions per milliliter, easily within the sensitivity of an RNA amplification architecture, but unattainable by antibody-based architectures that simply target viral antigens. Here, we describe such a test based on a displaceable probe loop amplification architecture.


Asunto(s)
/métodos , Portador Sano/virología , /aislamiento & purificación , Adulto , Antígenos Virales/genética , /epidemiología , Portador Sano/diagnóstico , Femenino , Humanos , Masculino , Técnicas de Amplificación de Ácido Nucleico/métodos , Pandemias , ARN Viral/genética , Sensibilidad y Especificidad , Carga Viral
18.
J Clin Virol ; 136: 104764, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33636553

RESUMEN

The current scale of public and private testing cannot be expected to meet the emerging need for higher levels of community-level and repeated screening of asymptomatic Canadians for SARS-CoV-2. Rapid point-of-care techniques are increasingly being offered to fill the gap in screening levels required to identify undiagnosed individuals with high viral loads. However, rapid, point-of-care tests often have lower sensitivity in practice. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 has proven sensitive and specific and provides visual results in minutes. Using a commercially available kit for RT-LAMP and primer set targetting nucleocapsid (N), we tested a blinded set of 101 archived nasopharyngeal (NP) swab samples with known RT-PCR results. RT-LAMP reactions were incubated at 65 °C for 30 min, using heat-inactivated nasopharyngeal swab sample in viral transport medium, diluted tenfold in water, as input. RT-LAMP agreed with all RT-PCR defined negatives (N = 51), and all positives with cycle threshold (Ct) less than 20 (N = 24), 65% of positives with Ct between 20-30 (N = 17), and no positives with Ct greater than 30 (N = 9). RT-LAMP requires fewer and different core components, so may not compete directly with the mainline testing workflow, preserving precious central laboratory resources for those with the greatest need. Careful messaging must be provided when using less-sensitive tests, so that people are not falsely reassured by negative results, but this caveat must be weighed against the clear benefits of reliably identifying those with high levels of virus in prioritized samples at the point of care.


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Enfermedades Asintomáticas , Canadá , Humanos , Tamizaje Masivo/métodos , Nasofaringe/virología , ARN Viral/análisis , Sensibilidad y Especificidad
19.
J Virol Methods ; 290: 114083, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33515664

RESUMEN

In the current pandemic of SARS-CoV-2, rapid identification of infected individuals is crucial for management and control of the outbreak. However, transport of samples, sample processing and RT-qPCR analysis in laboratories are time-consuming. Here we present a prototype of a novel nucleic acid-based test format - pulse controlled amplification - that allows detection of SARS-CoV-2 directly from up to eight swab samples simultaneously without the need for RNA extraction within 25 min with a sensitivity of 100 % for samples with a viral load of ≥ 1.6 × 10e3 copies/µl This new principle might pave the way to rapid and sensitive point of care testing.


Asunto(s)
/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /aislamiento & purificación , /normas , Humanos , Técnicas de Amplificación de Ácido Nucleico/normas , Pruebas en el Punto de Atención , ARN Viral/genética , Sensibilidad y Especificidad
20.
Biosensors (Basel) ; 11(2)2021 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-33513845

RESUMEN

Worldwide infection disease due to SARS-CoV-2 is tremendously affecting our daily lives. High-throughput detection methods for nucleic acids are emergently desired. Here, we show high-sensitivity and high-throughput metasurface fluorescence biosensors that are applicable for nucleic acid targets. The all-dielectric metasurface biosensors comprise silicon-on-insulator nanorod array and have prominent electromagnetic resonances enhancing fluorescence emission. For proof-of-concept experiment on the metasurface biosensors, we have conducted fluorescence detection of single-strand oligoDNAs, which model the partial sequences of SARS-CoV-2 RNA indicated by national infection institutes, and succeeded in the high-throughput detection at low concentrations on the order of 100 amol/mL without any amplification technique. As a direct detection method, the metasurface fluorescence biosensors exhibit high performance.


Asunto(s)
Técnicas Biosensibles/métodos , /diagnóstico , /genética , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/análisis , Sensibilidad y Especificidad
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...