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1.
Sensors (Basel) ; 21(9)2021 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-33946302

RESUMEN

Electrowetting-on-dielectric (EWOD) is a microfluidic technology used for manipulating liquid droplets at microliter to nanoliter scale. EWOD has the ability to facilitate the accurate manipulation of liquid droplets, i.e., transporting, dispensing, splitting, and mixing. In this work, EWOD fabrication with suitable and affordable materials is proposed for creating EWOD lab-on-a-chip platforms. The EWOD platforms are applied for the diagnosis of early mortality syndrome (EMS) in shrimp by utilizing the colorimetric loop-mediated isothermal amplification method with pH-sensitive xylenol orange (LAMP-XO) diagnosis technique. The qualitative sensitivity is observed by comparing the limit of detection (LOD) while performing the LAMP-XO diagnosis test on the proposed lab-on-a-chip EWOD platform, alongside standard LAMP laboratory tests. The comparison results confirm the reliability of EMS diagnosis on the EWOD platform with qualitative sensitivity for detecting the EMS DNA plasmid concentration at 102 copies in a similar manner to the common LAMP diagnosis tests.


Asunto(s)
Electrohumectación , Técnicas Analíticas Microfluídicas , Colorimetría , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Reproducibilidad de los Resultados
2.
Int J Mol Med ; 47(6)2021 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-33846767

RESUMEN

The Coronavirus Disease 2019 (COVID­19) pandemic has forced the scientific community to rapidly develop highly reliable diagnostic methods in order to effectively and accurately diagnose this pathology, thus limiting the spread of infection. Although the structural and molecular characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) were initially unknown, various diagnostic strategies useful for making a correct diagnosis of COVID­19 have been rapidly developed by private research laboratories and biomedical companies. At present, rapid antigen or antibody tests, immunoenzymatic serological tests and molecular tests based on RT­PCR are the most widely used and validated techniques worldwide. Apart from these conventional methods, other techniques, including isothermal nucleic acid amplification techniques, clusters of regularly interspaced short palindromic repeats/Cas (CRISPR/Cas)­based approaches or digital PCR methods are currently used in research contexts or are awaiting approval for diagnostic use by competent authorities. In order to provide guidance for the correct use of COVID­19 diagnostic tests, the present review describes the diagnostic strategies available which may be used for the diagnosis of COVID­19 infection in both clinical and research settings. In particular, the technical and instrumental characteristics of the diagnostic methods used are described herein. In addition, updated and detailed information about the type of sample, the modality and the timing of use of specific tests are also discussed.


Asunto(s)
/métodos , /diagnóstico , Animales , Técnicas Biosensibles/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Invenciones , Microscopía Electrónica/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /aislamiento & purificación , Cultivo de Virus/métodos
3.
Lancet ; 397(10282): 1351-1362, 2021 04 10.
Artículo en Inglés | MEDLINE | ID: mdl-33798499

RESUMEN

BACKGROUND: A new variant of SARS-CoV-2, B.1.1.7, emerged as the dominant cause of COVID-19 disease in the UK from November, 2020. We report a post-hoc analysis of the efficacy of the adenoviral vector vaccine, ChAdOx1 nCoV-19 (AZD1222), against this variant. METHODS: Volunteers (aged ≥18 years) who were enrolled in phase 2/3 vaccine efficacy studies in the UK, and who were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 or a meningococcal conjugate control (MenACWY) vaccine, provided upper airway swabs on a weekly basis and also if they developed symptoms of COVID-19 disease (a cough, a fever of 37·8°C or higher, shortness of breath, anosmia, or ageusia). Swabs were tested by nucleic acid amplification test (NAAT) for SARS-CoV-2 and positive samples were sequenced through the COVID-19 Genomics UK consortium. Neutralising antibody responses were measured using a live-virus microneutralisation assay against the B.1.1.7 lineage and a canonical non-B.1.1.7 lineage (Victoria). The efficacy analysis included symptomatic COVID-19 in seronegative participants with a NAAT positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to vaccine received. Vaccine efficacy was calculated as 1 - relative risk (ChAdOx1 nCoV-19 vs MenACWY groups) derived from a robust Poisson regression model. This study is continuing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Participants in efficacy cohorts were recruited between May 31 and Nov 13, 2020, and received booster doses between Aug 3 and Dec 30, 2020. Of 8534 participants in the primary efficacy cohort, 6636 (78%) were aged 18-55 years and 5065 (59%) were female. Between Oct 1, 2020, and Jan 14, 2021, 520 participants developed SARS-CoV-2 infection. 1466 NAAT positive nose and throat swabs were collected from these participants during the trial. Of these, 401 swabs from 311 participants were successfully sequenced. Laboratory virus neutralisation activity by vaccine-induced antibodies was lower against the B.1.1.7 variant than against the Victoria lineage (geometric mean ratio 8·9, 95% CI 7·2-11·0). Clinical vaccine efficacy against symptomatic NAAT positive infection was 70·4% (95% CI 43·6-84·5) for B.1.1.7 and 81·5% (67·9-89·4) for non-B.1.1.7 lineages. INTERPRETATION: ChAdOx1 nCoV-19 showed reduced neutralisation activity against the B.1.1.7 variant compared with a non-B.1.1.7 variant in vitro, but the vaccine showed efficacy against the B.1.1.7 variant of SARS-CoV-2. FUNDING: UK Research and Innovation, National Institute for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.


Asunto(s)
Anticuerpos Neutralizantes/sangre , /prevención & control , /inmunología , Adolescente , Adulto , /efectos adversos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Amplificación de Ácido Nucleico , Pandemias/prevención & control , Método Simple Ciego , Reino Unido/epidemiología , Carga Viral , Adulto Joven
4.
J Biomed Nanotechnol ; 17(3): 407-415, 2021 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-33875075

RESUMEN

Portable nucleic acid detection (PNAD) systems are performed for sample processing, amplification and detection automatically in an individual device realizing "sample in, answer out." For this goal, numerous function modules should be integrated in a diminutive device, in which temperature controller is one of the most important modules. In a nucleic acid detection process, both sample processing and polymerase chain reaction (PCR) require fast and accurate temperature control to increase concentration and purity of the extraction product and to improve amplification efficiency. In this paper, a dual-channel temperature controller for PNAD systems is developed, which contains a printed circuit board (PCB) and an integrated control program with a fast and accurate control strategy. According to the principle of nucleic acid detection based on magnetic nanoparticles, the controller can work in different modes such as high-precision heating control for nucleic acid extraction, rapid thermal cycle control for PCR, and rate adjustable constant heating/cooling control for melting curve. Evaluatively, the average heating/cooling rate of the module can exceed about 6 C/s, while the temperature fluctuation was less than ± 0.1°C, which can meet the demands of PNAD systems very well.


Asunto(s)
Nanopartículas de Magnetita , Ácidos Nucleicos , Calefacción , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Temperatura
5.
Sensors (Basel) ; 21(5)2021 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-33802488

RESUMEN

Isothermal amplification techniques are emerging nowadays for the rapid and accurate detection of pathogenic bacteria in low resource settings, where many infectious diseases are endemic, and the lack of reliable power supply, trained personnel and specialized facilities pose critical barriers for timely diagnosis. This work addresses the detection of E. coli based on DNA isothermal amplification performed on magnetic particles (MPs) followed by electrochemical genosensing on disposable electrodes by square-wave voltammetry. In this approach, the bacterial DNA is preconcentrated using a target-specific magnetic probe and then amplified on the MPs by rolling circle amplification (RCA). Two different electrochemical readout methods for the RCA amplicons are tested. The first one relied on the labelling of the magnetic RCA product with a digoxigenin probe followed by the incubation with antiDIG-HRP antibody as electrochemical reporter. In the second case, the direct detection with an HRP-probe was performed. This latter strategy showed an improved analytical performance, while simultaneously avoiding the use of thermocyclers or bulky bench top equipment.


Asunto(s)
Escherichia coli , Técnicas de Amplificación de Ácido Nucleico , ADN Bacteriano/genética , Técnicas Electroquímicas , Escherichia coli/genética
6.
Medicine (Baltimore) ; 100(17): e25632, 2021 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-33907119

RESUMEN

ABSTRACT: The 17 Provincial Institutes of Health and Environment (PIHEs) in Korea use HIV antibody, antigen, and Western blot assays for confirmatory testing of HIV infection. The Korea Disease Control and Prevention Agency (KDCA) has further included p24 antigen neutralization and nucleic acid tests (NATs) since 2015. Our study aimed to investigate the effect of this new testing algorithm on the confirmation rate of HIV infection.Annual changes, from 2012 through 2017, in positive or indeterminate HIV confirmatory results were compared for the two algorithms between the PIHEs and the KDCA. Fiebig stages and Western blot p31 band were used to identify the diagnostic proportions of acute or early chronic HIV for the two algorithms.The number of positive cases in the samples requested from PIHEs for reconfirmation by the KDCA has steadily increased from 10.3% in 2014 to 33.3% in 2017. However, the number of indeterminate cases dropped sharply, from 71.9% in 2014 to 14.0% in 2017. The results for the p31 reactive band were 27.4% and 88.4% for the KDCA and PIHEs, respectively. Of positive cases reported by the KDCA, 22.9% were in the early acute stage and Fiebig stages I to II.The new testing algorithm has improved the diagnosis of HIV infections in the early acute stage. Early confirmatory diagnosis can prevent secondary transmission of HIV and provide early treatment opportunities for people living with HIV infection.


Asunto(s)
Algoritmos , Western Blotting/estadística & datos numéricos , Infecciones por VIH/diagnóstico , Inmunoensayo/estadística & datos numéricos , Técnicas de Amplificación de Ácido Nucleico/estadística & datos numéricos , Diagnóstico Precoz , VIH/inmunología , Anticuerpos Anti-VIH/análisis , Antígenos VIH/análisis , Infecciones por VIH/epidemiología , Humanos , República de Corea/epidemiología , Sensibilidad y Especificidad
7.
Elife ; 102021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33876726

RESUMEN

Diagnosis of SARS-CoV-2 (COVID-19) requires confirmation by reverse transcription-polymerase chain reaction (RT-PCR). Abbott ID NOW provides fast results but has been criticized for low sensitivity. Here we determine the sensitivity of ID NOW in an ambulatory population presented for testing. The study enrolled 785 symptomatic patients, of whom 21 were positive by both ID NOW and RT-PCR, and 2 only by RT-PCR. All 189 asymptomatic patients tested negative. The positive percent agreement between the ID NOW assay and the RT-PCR assay was 91.3%, and negative percent agreement was 100%. The results from the current study were included into a larger systematic review of literature where at least 20 subjects were simultaneously tested using ID NOW and RT-PCR. The overall sensitivity for ID NOW assay was calculated at 84% (95% confidence interval 55-96%) and had the highest correlation to RT-PCR at viral loads most likely to be associated with transmissible infections.


Asunto(s)
/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /aislamiento & purificación , Adulto , Femenino , Genoma Viral , Humanos , Masculino , Persona de Mediana Edad , Nariz/virología , Técnicas de Amplificación de Ácido Nucleico/métodos , Sistemas de Atención de Punto , Sensibilidad y Especificidad , Manejo de Especímenes , Carga Viral
8.
Anal Chim Acta ; 1158: 338390, 2021 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-33863409

RESUMEN

The development of rapid, highly sensitive, and selective methods for the diagnosis of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should help to prevent the spread of this pandemic virus. In this study, we combined recombinase polymerase amplification (RPA), as a means of isothermal DNA amplification, with an rkDNA-graphene oxide (GO) probe system to allow the rapid detection of SARS-CoV-2 with high sensitivity and selectivity. We used in situ enzymatic synthesis to prepare an rkDNA probe that was complementary to an RPA-amplified sequence of the target N-gene of SARS-CoV-2. The fluorescence of this rkDNA was perfectly quenched in the presence of GO. When the quenched rkDNA-GO system was added to the RPA-amplified sequence of the target SARS-CoV-2, the fluorescence recovered dramatically. The combined RPA/rkDNA-GO system exhibited extremely high selectivity (discrimination factor: 17.2) and sensitivity (LOD = 6.0 aM) for the detection of SARS-CoV-2. The total processing time was only 1.6 h. This combined RPA/rkDNA-GO system appears to be a very efficient and simple method for the point-of-care detection of SARS-CoV-2.


Asunto(s)
/diagnóstico , Grafito , Técnicas de Amplificación de Ácido Nucleico , /aislamiento & purificación , Humanos , Pandemias , Recombinasas , Sensibilidad y Especificidad
9.
Acta Virol ; 65(1): 27-32, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33827220

RESUMEN

Marek's disease (MD) is a lymphoproliferative disease of chickens with strong economic impact on poultry industry. Although successful vaccination has enabled control of the disease, outbreaks occur in commercial flocks, resulting in substantial economic losses. Together with vaccination, accurate and fast diagnosis of MD remain the most important tools for its efficient control. MD diagnosis currently relies mainly on the identification of its causative agent, Marek's disease virus type 1 (MDV-1). Nucleic acid amplification techniques have been successfully applied for identification of MDV DNA in field samples and also for studies of virus-host interactions. In this review we want to summarize recent advances in the development of standard and quantitative PCR techniques and their use in rapid MD diagnosis, including differentiation of pathogenic and vaccine MD viruses. PCR protocols have served as a useful tool for clarification of processes associated with MDV infection in chickens, such as virus spread and release, and effect of vaccine virus on progress of MD. Here, we also describe a novel multi-species qPCR methodology for identification and quantification of MDV DNA, enabling its detection in various bird species that are the most susceptible to MDV infection. Keywords: Marek's disease; MDV; diagnosis; nucleic acid detection; duplex quantitative PCR.


Asunto(s)
Herpesvirus Gallináceo 2 , Enfermedad de Marek , Ácidos Nucleicos , Enfermedades de las Aves de Corral , Animales , Pollos , Herpesvirus Gallináceo 2/genética , Enfermedad de Marek/diagnóstico , Técnicas de Amplificación de Ácido Nucleico , Enfermedades de las Aves de Corral/diagnóstico
10.
Biotechniques ; 70(4): 218-225, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33820475

RESUMEN

Evaluation of the performance of a new set of primers defined from the ORF1ab sequence, and its combination with a previously published set of primers from the N sequence, to detect SARS-CoV-2 RNA by the loop-mediated isothermal amplification technique is presented. The ORF1ab primer set enables visual detection of SARS-CoV-2 RNA in 16 min. In addition, a simultaneous reaction with both ORF1ab and N primers allows for higher sensitivity of detection, particularly when low numbers of copies are present (250 viral RNA copies). Further, the protocol is able to detect viral RNA in saliva samples. The procedure reported could be easily implemented in the generation of a new and sensitive rapid point-of care device for SARS-CoV-2 RNA visual detection.


Asunto(s)
/métodos , Colorimetría/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /genética , /virología , Cartilla de ADN , Humanos , Poliproteínas/genética , Proteínas Virales/genética
11.
Artículo en Inglés | MEDLINE | ID: mdl-33802332

RESUMEN

Background: Health care systems in the United States are continuously expanding and contracting spaces to treat patients with coronavirus disease 2019 (COVID-19) in intensive care units (ICUs). As a result, hospitals must effectively decontaminate and contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in constructed and deconstructed ICUs that care for patients with COVID-19. We assessed decontamination of a COVID-19 ICU and examined the containment efficacy of combined contact and droplet precautions in creating and maintaining a SARS-CoV-2-negative ICU "antechamber". Methods: To examine the efficacy of chemical decontamination, we used high-density, semi-quantitative environmental sampling to detect SARS-CoV-2 on surfaces in a COVID-19 ICU and COVID-19 ICU antechamber. Quantitative real-time polymerase chain reaction was used to measure viral RNA on surfaces. Viral location mapping revealed the distribution of viral RNA in the COVID-19 ICU and COVID-19 ICU antechamber. Results were further assessed using loop-mediated isothermal amplification. Results: We collected 224 surface samples pre-decontamination and 193 samples post-decontamination from a COVID-19 ICU and adjoining COVID-19 ICU antechamber. We found that 46% of antechamber objects were positive for SARS-CoV-2 pre-decontamination despite the construction of a swinging door barrier system, implementation of contact precautions, and installation of high-efficiency particulate air filters. The object positivity rate reduced to 32.1% and viral particle rate reduced by 95.4% following decontamination. Matched items had an average of 432.2 ± 2729 viral copies/cm2 pre-decontamination and 19.2 ± 118 viral copies/cm2 post-decontamination, demonstrating significantly reduced viral surface distribution (p < 0.0001). Conclusions: Environmental sampling is an effective method for evaluating decontamination protocols and validating measures used to contain SARS-CoV-2 viral particles. While chemical decontamination effectively removes detectable viral RNA from surfaces, our approach to droplet/contact containment with an antechamber was not highly effective. These data suggest that hospitals should plan for the potential of aerosolized virions when creating strategies to contain SARS-CoV-2.


Asunto(s)
Descontaminación , Humanos , Unidades de Cuidados Intensivos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico
12.
Sensors (Basel) ; 21(9)2021 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-33925730

RESUMEN

Nucleic acid (NA) extraction is a basic step for genetic analysis, from scientific research to diagnostic and forensic applications. It aims at preparing samples for its application with biomolecular technologies such as isothermal and non-isothermal amplification, hybridization, electrophoresis, Sanger sequencing and next-generation sequencing. Multiple steps are involved in NA collection from raw samples, including cell separation from the rest of the specimen, cell lysis, NA isolation and release. Typically, this process needs molecular biology facilities, specialized instrumentation and labor-intensive operations. Microfluidic devices have been developed to analyze NA samples with high efficacy and sensitivity. In this context, the integration within the chip of the sample preparation phase is crucial to leverage the promise of portable, fast, user-friendly and economic point-of-care solutions. This review presents an overview of existing lab-on-a-chip (LOC) solutions designed to provide automated NA extraction from human raw biological fluids, such as whole blood, excreta (urine and feces), saliva. It mainly focuses on LOC implementation aspects, aiming to describe a detailed panorama of strategies implemented for different human raw sample preparations.


Asunto(s)
Técnicas Analíticas Microfluídicas , Ácidos Nucleicos , Humanos , Dispositivos Laboratorio en un Chip , Microfluídica , Técnicas de Amplificación de Ácido Nucleico , Sistemas de Atención de Punto
13.
Med J (Ft Sam Houst Tex) ; (PB 8-21-01/02/03): 37-49, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33666911

RESUMEN

SARS-CoV-2 has highlighted the requirement for a drastic change in pandemic response. While cases continue to rise, there is an urgent need to deploy sensitive and rapid testing in order to identify potential outbreaks before there is an opportunity for further community spread. Currently, reverse transcription quantitative polymerase chain reaction (RT-qPCR) is considered the gold standard for diagnosing an active infection, using a nasopharyngeal swab; however, it can take days after symptoms develop to properly identify and trace the infection. While many civilian jobs can be performed remotely, the Department of Defense (DOD) is by nature a very fluid organization which requires in-person interaction and a physical presence to maintain effectiveness. In this commentary, we examine several current and emergent technologies and their ability to identify both active and previous SARS-CoV-2 infection, possibly in those without symptoms. Further, we will explore an ongoing study at the Air Force Research Laboratory, utilizing Reverse Transcription Loop-mediated isothermal amplification (RT-LAMP), next-generation sequencing, and the presence of SARS-CoV-2 antibodies through Lateral Flow Immunoassays. The ability to identify SARS-CoV-2 through volatile organic compound biomarker identification will also be explored. By exploring and validating multiple testing strategies, and contributing to Operation Warp Speed, the DOD is postured to respond to SARS-CoV-2, and future pandemics.


Asunto(s)
/diagnóstico , Personal Militar , /aislamiento & purificación , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Estados Unidos
14.
Anal Chim Acta ; 1154: 338310, 2021 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-33736798

RESUMEN

Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 min with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per µL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.


Asunto(s)
/métodos , /diagnóstico , /virología , Cartilla de ADN/química , Cartilla de ADN/metabolismo , Humanos , Límite de Detección , Técnicas de Diagnóstico Molecular , Nasofaringe/virología , Técnicas de Amplificación de Ácido Nucleico , ARN Viral/análisis , ARN Viral/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , /aislamiento & purificación , Factores de Tiempo
15.
Front Public Health ; 9: 568603, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33748054

RESUMEN

The declaration of COVID-19 as a global pandemic has warranted the urgent need for technologies and tools to be deployed for confirming diagnosis of suspected cases. Diagnostic testing for COVID-19 is critical for understanding epidemiology, contract-tracing, case management, and to repress the transmission of the SARS-CoV-2. Currently, the Nucleic Acid Amplification Test (NAAT)-based RT-PCR technique is a gold standard test used for routine diagnosis of COVID-19 infection. While there are many commercially available RT-PCR assay kits available in the market, selection of highly sensitive, specific, and validated assays is most crucial for the accurate diagnosis of COVID-19 infection. Laboratory diagnosis of SARS-CoV-2 is extremely important in the disease and outbreak management. Development of rapid point of care tests with better sensitivity and specificity is the critical need of the hour as this will help accurate diagnosis and aid in containing the spread of SARS-CoV-2 infection. Early detection of viral infection greatly enhances implementation of specific public health intervention, such as infection control, environmental decontamination, and the closure of specific high-risk zones. Large-scale sequencing of SARS-CoV-2 genome isolated from affected populations across the world needs to be carried to monitor mutations that might affect performance of molecular tests. Creation of genome repositories and open-source genetic databases for use by global researchers is clearly the way forward to manage COVID-19 outbreak and accelerate vaccine development. This review summarizes various molecular diagnostics methods, technical guidelines, and advanced testing strategies adopted in India for laboratory diagnosis of COVID-19.


Asunto(s)
/normas , Técnicas de Laboratorio Clínico , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico/normas , /prevención & control , Humanos , India , Control de Infecciones , Mutación/genética , Pruebas en el Punto de Atención
16.
Nat Commun ; 12(1): 1660, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712587

RESUMEN

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin-angiotensin-aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.


Asunto(s)
/genética , /genética , Adulto , Anciano , Antagonistas de Receptores de Angiotensina/farmacología , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Antivirales/farmacología , /epidemiología , Interacciones Farmacológicas , Femenino , Perfilación de la Expresión Génica , Genoma Viral , Antígenos HLA/genética , Interacciones Microbiota-Huesped/efectos de los fármacos , Interacciones Microbiota-Huesped/genética , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Ciudad de Nueva York/epidemiología , Técnicas de Amplificación de Ácido Nucleico , Pandemias , RNA-Seq , /efectos de los fármacos
17.
ACS Sens ; 6(3): 881-888, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33645226

RESUMEN

Coronavirus Disease 2019 (COVID-19), which is caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), has rapidly spread leading to a global pandemic. Here, we combined multiple cross displacement amplification (MCDA) with CRISPR-Cas12a-based detection to develop a novel diagnostic test (MCCD) and applied for the diagnosis of COVID-19, called COVID-19 MCCD. The MCCD protocol conducts reverse transcription MCDA (RT-MCDA) reaction for RNA templates followed by CRISPR-Cas12a/CrRNA complex detection of predefined target sequences after which degradation of a single-strand DNA (ssDNA) molecule confirms detection of the target sequence. Two MCDA primer sets and two CrRNAs were designed targeting the opening reading frame 1a/b (ORF1ab) and nucleoprotein (N) of SARS-CoV-2. The optimal conditions include two RT-MCDA reactions at 63 °C for 35 min and a CRISPR-Cas12a/CrRNA detection reaction at 37 °C for 5 min. The COVID-19 MCCD assay can be visualized on a lateral flow biosensor (LFB) and completed within 1 h including RNA extraction (15 min), RT-MCDA reaction (35 min), CRISPR-Cas12a/CrRNA detection reaction (5 min), and reporting of result (within 2 min). The COVID-19 MCCD assay is very sensitive and detects the target gene with as low as seven copies per test and does not cross-react with non-SARS-CoV-2 templates. SARS-CoV-2 was detected in 37 of 37 COVID-19 patient samples, and nonpositive results were detected from 77 non-COVID-19 patients. Therefore, the COVID-19 MCCD assay is a useful tool for the reliable and quick diagnosis of SARS-CoV-2 infection.


Asunto(s)
Proteínas Bacterianas , Proteínas Asociadas a CRISPR , Sistemas CRISPR-Cas , Endodesoxirribonucleasas , Técnicas de Amplificación de Ácido Nucleico , ARN Viral , /genética , Técnicas Biosensibles , Humanos , Sensibilidad y Especificidad
18.
Food Chem ; 351: 129348, 2021 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-33647699

RESUMEN

Adulteration of food ingredients, particularly replacement of high-value milk with low-cost milk, affects food safety. For rapid and accurate identification of the possible adulterating milk species in an unknown sample, a centrifugal microfluidic chip-based real-time fluorescent multiplex loop-mediated isothermal amplification (LAMP) assay was developed to simultaneously detect milk from cow, camel, horse, goat, and yak. Using precoated primers in different reaction wells, the centrifugal microfluidic chip markedly simplified the detection process and reduced false-positive results. The entire amplification was completed within 90 min with a genomic detection limit of 0.05 ng/µL in cow, camel, horse, and goat milk and 0.005 ng/µL in yak milk. Using simulated adulterated samples for validation, the detection limit for adulterated milk samples was 2.5%, satisfying authentication requirements, as the proportion of adulterated milk higher than 10% affects economic interests. Therefore, this simple, centrifugal, microfluidic chip-based multiplex real-time fluorescent LAMP assay can simultaneously detect common milk species in commercial products to enable accurate labeling.


Asunto(s)
Centrifugación/instrumentación , Calidad de los Alimentos , Dispositivos Laboratorio en un Chip , Leche/química , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Animales , Bovinos , Cartilla de ADN/genética , Femenino , Leche/normas , Factores de Tiempo
19.
Biosens Bioelectron ; 182: 113168, 2021 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-33780853

RESUMEN

We report an electrochemical biosensor combined with recombinase polymerase amplification (RPA) for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2. The electrochemical biosensor based on a multi-microelectrode array allows the detection of multiple target genes by differential pulse voltammetry. The RPA reaction involves hybridization of the RPA amplicon with thiol-modified primers immobilized on the working electrodes, which leads to a reduction of current density as amplicons accumulate. The assay results in shorter "sample-to-answer" times than conventional PCR without expensive thermo-cycling equipment. The limits of detection are about 0.972 fg/µL (RdRP gene) and 3.925 fg/µL (N gene), which are slightly lower than or comparable to that of RPA assay results obtained by gel electrophoresis without post-amplification purification. The combination of electrochemical biosensors and the RPA assay is a rapid, sensitive, and convenient platform that can be potentially used as a point-of-care test for the diagnosis of COVID-19.


Asunto(s)
Técnicas Biosensibles , Técnicas de Amplificación de Ácido Nucleico , Pruebas en el Punto de Atención , Humanos , Sensibilidad y Especificidad
20.
Vopr Virusol ; 66(1): 17-28, 2021 03 07.
Artículo en Ruso | MEDLINE | ID: mdl-33683062

RESUMEN

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificación de Ácido Nucleico/normas , ARN Viral/genética , /genética , Artefactos , /normas , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Sondas de ADN/genética , Sondas de ADN/metabolismo , Humanos , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad
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