Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 6.284
Filtrar
1.
Talanta ; 225: 121898, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33592692

RESUMEN

The current situation of the Covid-19 pandemic is indicated by a huge number of infections, high lethality, and rapid spread. These circumstances have stopped the activity of almost the entire world, affecting severely the global economy. A rapid diagnosis of the Covid-19 and a generalized testing protocol is essential to fight against the pandemic and to maintain health control in the population. Principal biosensing and diagnostic technologies used to monitor the spread of the SARS-CoV-2 are based on specific genomic analysis and rapid immune tests, both with different technology platforms that include advantages and disadvantages. Most of the in vitro diagnosis companies are competing to be the first on validating under different regulations their technology for placing their platforms for Covid-19 detection as fast as possible in this big international market. A comprehensive analysis of the commercialized technologies for the genomic based sensing and the antibody/antigen detection methods devoted to Covid-19 diagnosis is described in this review, which have been detailed and listed under different countries regulations. The effectiveness of the described technologies throughout the different stages of the disease and a critical comparison of the emerging technologies in the market to counterattack this pandemic have been discussed.


Asunto(s)
/diagnóstico , Inmunoensayo/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , /aislamiento & purificación , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , /virología , Humanos , Pandemias , /fisiología , Sensibilidad y Especificidad
2.
PLoS One ; 16(2): e0246867, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33566873

RESUMEN

Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.


Asunto(s)
/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , ARN Viral/genética , /aislamiento & purificación , Humanos , Límite de Detección , Tamizaje Masivo , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Saliva/virología , Manejo de Especímenes , Factores de Tiempo
3.
Anal Methods ; 13(2): 169-178, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33399137

RESUMEN

We demonstrate a loop-mediated isothermal amplification (LAMP) method to detect and amplify SARS-CoV-2 genetic sequences using a set of in-house designed initiators that target regions encoding the N protein. We were able to detect and amplify SARS-CoV-2 nucleic acids in the range of 62 to 2 × 105 DNA copies by this straightforward method. Using synthetic SARS-CoV-2 samples and RNA extracts from patients, we demonstrate that colorimetric LAMP is a quantitative method comparable in diagnostic performance to RT-qPCR (i.e., sensitivity of 92.85% and specificity of 81.25% in a set of 44 RNA extracts from patients analyzed in a hospital setting).


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN/análisis , Carga Viral/métodos , /diagnóstico , Colorimetría/métodos , ADN/análisis , ADN/química , Colorantes Fluorescentes/química , Humanos , Sustancias Intercalantes/química , Fenolsulfonftaleína/química , Fosfoproteínas , ARN/química
4.
Sci Rep ; 11(1): 2261, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33500503

RESUMEN

The diagnosis of COVID-19 relies on the direct detection of SARS-CoV-2 RNA in respiratory specimens by RT-PCR. The pandemic spread of the disease caused an imbalance between demand and supply of materials and reagents needed for diagnostic purposes including swab sets. In a comparative effectiveness study, we conducted serial follow-up swabs in hospitalized laboratory-confirmed COVID-19 patients. We assessed the diagnostic performance of an in-house system developed according to recommendations by the US CDC. In a total of 96 serial swabs, we found significant differences in the accuracy of the different swab systems to generate a positive result in SARS-CoV-2 RT-PCR, ranging from around 50 to 80%. Of note, an in-house swab system was superior to most commercially available sets as reflected by significantly lower Ct values of viral genes. Thus, a simple combination of broadly available materials may enable diagnostic laboratories to bypass global limitations in the supply of swab sets.


Asunto(s)
/instrumentación , Equipos Desechables/provisión & distribución , Técnicas de Diagnóstico Molecular/instrumentación , /aislamiento & purificación , /métodos , Técnicas de Laboratorio Clínico , Pruebas Diagnósticas de Rutina , Genes Virales , Humanos , Técnicas de Diagnóstico Molecular/métodos , Control de Calidad , ARN Viral/análisis , Reproducibilidad de los Resultados , Asignación de Recursos , Manejo de Especímenes
5.
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
6.
Sci Rep ; 11(1): 2402, 2021 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-33510181

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
7.
Sci Rep ; 11(1): 1820, 2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33469065

RESUMEN

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.


Asunto(s)
/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /genética , /virología , Calor , Humanos , Nasofaringe/virología , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Inactivación de Virus
8.
Medicine (Baltimore) ; 100(1): e24062, 2021 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-33429770

RESUMEN

ABSTRACT: Colorectal cancer is a heterogeneous disease with multiple epigenetic alterations and different molecular features. The molecular classification is based on 2 major distinct pathways: microsatellite stable pathway and the microsatellite instability pathway. Molecular profiling of colorectal cancer provides important information regarding treatment and prognosis. Aim of the study was to assess the frequency of microsatellite instability in colon cancer and the clinicopathological characteristics of the tumors with high level of microsatellite instability (MSI-H) in our region. The secondary outcome was to assess the frequency of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in colon cancer.The study included 129 patients with colon cancer fit for surgery. Demographic data, clinical and pathological data, immunohistochemistry staining pattern (4 mismatch repair proteins were investigated), and BRAF gene mutations were assessed. According to microsatellite instability status by polymerase chain reaction, patients were divided into 3 groups: microsatellite stable (MSS) = 108 patients, high level of microsatellite instability (MSI-H) = 15 patients and low level of microsatellite instability (MSI-L) = 6 patients. Different clinicopathological comparisons between MSS and MSI-H patients, and between MSS and MSI-L patients were performed.Microsatellite instability was found in 16.3% patients: 11.6% had MSI-H and 4.7% had MSI-L. Significantly more patients in the MSI-H group than in the MSS group were female (P = .01) and had a family history of colon cancer (P < .001). MSI-H and MSI-L groups were associated with the ascending colon location of the tumors, were mostly type G3, T2, and stage I whereas MSS tumors were mostly G2, pT3, and stage III. Overall, BRAF mutations were identified in 18/129 patients (13.9%). BRAF mutant tumors were predominantly associated with MSI-H and MSI-L tumors. Immunohistochemistry had a sensitivity of 76% and a specificity of 89% in detecting MSI tumors and an accuracy of 87.6%.The frequency of microsatellite instability in our study was 16.3%. MSI-H is a distinct molecular phenotype of colon cancer with particular features: female gender, family history of colorectal cancer, a predilection for the ascending colon, poorly differentiated, predominantly T2, and stage I. The frequency of BRAF mutations was 13.9% and mutations were more often present in the MSI tumors.


Asunto(s)
Neoplasias del Colon/genética , Técnicas de Diagnóstico Molecular/métodos , Anciano , Neoplasias del Colon/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/análisis , Proteínas Proto-Oncogénicas B-raf/genética , Rumanía/epidemiología , Estadísticas no Paramétricas
9.
Sci Rep ; 11(1): 1873, 2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33479389

RESUMEN

The development of alternative isothermal amplification assays including multiple cross displacement amplification (MCDA) may address speed and portability limitations of real-time PCR (rt-PCR) methods for SARS-CoV-2 detection. We developed a novel SARS-CoV-2 MCDA assay and compared its speed and sensitivity to loop-mediated isothermal amplification (LAMP) and rt-PCR. Two MCDA assays targeting SARS-CoV-2 N gene and ORF1ab were designed. The fastest time to detection and sensitivity of MCDA was compared to LAMP and rt-PCR using DNA standards and transcribed RNA. For the N gene, MCDA was faster than LAMP and rt-PCR by 10 and 20 min, respectively with fastest time to detection at 5.2 min. rt-PCR had the highest sensitivity with the limit of detection at 10 copies/µl compared with MCDA (100 copies/µl) and LAMP (500 copies/µl). For ORF1ab, MCDA and LAMP had similar speed with fastest time to detection at 9.7 and 8.4 min, respectively. LAMP was more sensitive for ORF1ab detection with 50 copies/µl compared to MCDA (500 copies/µl). In conclusion, different nucleic acid amplification methods provide different advantages. MCDA is the fastest nucleic acid amplification method for SARS-CoV-2 while rt-PCR is the most sensitive. These advantages should be considered when determining the most suitable nucleic acid amplification methods for different applications.


Asunto(s)
/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , /aislamiento & purificación , Bioensayo/métodos , /métodos , Técnicas de Laboratorio Clínico/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Fosfoproteínas/genética , Poliproteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Proteínas Virales/genética
10.
Biosens Bioelectron ; 177: 113005, 2021 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-33486135

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
11.
J Clin Virol ; 135: 104720, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33418142

RESUMEN

BACKGROUND: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit components, buffers and even plastics has resulted in suboptimal testing procedures worldwide. Alternative workflows have been implemented to overcome these difficulties. Recently a liquid based sample prep has been launched as solution to overcome limitations in relation to nucleic acid extraction. OBJECTIVE: Multicenter evaluation of the QIAprep& Viral RNA UM kit (QIA P&A) for rapid sample preparation and real-time PCR detection of SARS-CoV-2 in comparison to standardized laboratory testing methods. STUDY DESIGN: Selected samples of the routine diagnostic workflow at Clinical Microbiology Laboratories of four Dutch hospitals have been subjected to the rapid QIA P&A protocol and the results have been compared to routine diagnostic data. RESULTS: Combining results of manual and automated procedures, a total of 377 clinical samples of which 202 had been tested positive with a wide range of CT values, showed almost complete concordance in the QIA P&A assay for samples up to CT values of 33 with one exception of CT 31. Prospectively 60 samples were tested and also showed 100 % concordance with 5 positives. The method has been automated by two centres. CONCLUSIONS: Despite an input of only 8 µL of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic.


Asunto(s)
/métodos , /virología , Tamizaje Masivo/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /genética , Técnicas de Laboratorio Clínico/métodos , Humanos , Técnicas de Diagnóstico Molecular/métodos , Pandemias/prevención & control , Estudios Prospectivos , Manejo de Especímenes/métodos , Flujo de Trabajo
12.
J Clin Virol ; 135: 104737, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33497932

RESUMEN

The GenMark Dx ePlex Respiratory Pathogen Panel (RP) is a multiplexed nucleic acid test for the qualitative detection of common viral and a few bacterial causes of respiratory tract infections. The ePlex RP has received FDA clearance for nasopharyngeal swab (NPS) specimens collected in viral transport media. In this study, we evaluated the performance of the ePlex RP panel in comparison to the NxTAG Respiratory Pathogen Panel (NxTAG-RPP) from Luminex in use in our laboratory, not only for NPS but also for bronchoalveolar lavage specimens (BAL). We also evaluated the impact of implementing the ePlex RP on the test turn-around time (TAT). The newest panel from GenMark Dx, the ePlex Respiratory Pathogen Panel 2 (RP2), which added the SARS-CoV-2 target to the RP was also evaluated for NPS. Verification of the performance of the ePlex RP for both NPS and BAL showed 93.3 % and 84.9 % total agreement with the NxTAG-RPP respectively. An overall comparison of the TAT after implementing the ePlex RP as compared to the NxTAG-RPP assay showed an average decrease of almost seven-fold.


Asunto(s)
Pruebas Diagnósticas de Rutina/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones del Sistema Respiratorio/diagnóstico , Lavado Broncoalveolar/métodos , Humanos , Nasofaringe/microbiología , Nasofaringe/virología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , /genética
13.
Int J Mol Sci ; 22(3)2021 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-33498408

RESUMEN

The COVID-19 pandemic caused by the SARS-CoV-2 virus, which first emerged in December 2019, represents an ongoing global public health emergency. Here, we developed an improved and highly sensitive approach to SARS-CoV-2 detection via coupling bioluminescence in real-time (BART) and reverse-transcriptase loop-mediated amplification (RT-LAMP) protocols (RT-LAMP-BART) and was also compatible with a digital LAMP system (Rainsuit), which did not allow for real-time quantification but did, nonetheless, facilitate absolute quantification with a comparable detection limit of 104 copies/mL. Through improving RNA availability in samples to ensure the target RNA present in reaction, we additionally developed a simulated digital RT-LAMP approach using this same principle to enlarge the overall reaction volume and to achieve real-time detection with a limit of detection of 10 copies/mL, and with further improvements in the overall dynamic range of this assay system being achieved through additional optimization.


Asunto(s)
/virología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas Virales/genética , Humanos , Límite de Detección , Mediciones Luminiscentes/métodos , Poliproteínas/genética , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transcripción Reversa
14.
Biosens Bioelectron ; 172: 112766, 2021 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-33126177

RESUMEN

The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.


Asunto(s)
/métodos , Sistemas CRISPR-Cas , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , /genética , Secuencia de Bases , /instrumentación , Humanos , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Técnicas de Amplificación de Ácido Nucleico/estadística & datos numéricos , Pandemias , ARN Viral/genética , Sensibilidad y Especificidad
16.
J Virol Methods ; 289: 114048, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33358911

RESUMEN

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Saliva/virología , Manejo de Especímenes/métodos , Humanos , ARN Viral/análisis
17.
Viruses ; 12(12)2020 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-33334037

RESUMEN

Recent outbreaks of African swine fever virus (ASFV) have seen the movement of this virus into multiple new regions with devastating impact. Many of these outbreaks are occurring in remote, or resource-limited areas, that do not have access to molecular laboratories. Loop-mediated isothermal amplification (LAMP) is a rapid point of care test that can overcome a range of inhibitors. We outline further development of a real-time ASFV LAMP, including field verification during an outbreak in Timor-Leste. To increase field applicability, the extraction step was removed and an internal amplification control (IAC) was implemented. Assay performance was assessed in six different sample matrices and verified for a range of clinical samples. A LAMP detection limit of 400 copies/rxn was determined based on synthetic positive control spikes. A colourmetric LAMP assay was also assessed on serum samples. Comparison of the LAMP assay to a quantitative polymerase chain reaction (qPCR) was performed on clinical ASFV samples, using both serum and oral/rectal swabs, with a substantial level of agreement observed. The further verification of the ASFV LAMP assay, removal of extraction step, implementation of an IAC and the assessment of a range of sample matrix, further support the use of this assay for rapid in-field detection of ASFV.


Asunto(s)
Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/virología , Brotes de Enfermedades , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Fiebre Porcina Africana/diagnóstico , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Femenino , Masculino , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/normas , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Porcinos , Viremia
18.
BMC Infect Dis ; 20(1): 947, 2020 Dec 11.
Artículo en Inglés | MEDLINE | ID: mdl-33308203

RESUMEN

BACKGROUND: Early detection of Zika virus (ZIKV) infection during the viremia and viruria facilitates proper patient management and mosquito control measurement to prevent disease spread. Therefore, a cost-effective nucleic acid detection method for the diagnosis of ZIKV infection, especially in resource-deficient settings, is highly required. METHODS: In the present study, a single-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the detection of both the Asian and African-lineage ZIKV. The detection limit, strain coverage and cross-reactivity of the ZIKV RT-LAMP assay was evaluated. The sensitivity and specificity of the RT-LAMP were also evaluated using a total of 24 simulated clinical samples. The ZIKV quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was used as the reference assay. RESULTS: The detection limit of the RT-LAMP assay was 3.73 ZIKV RNA copies (probit analysis, P ≤ 0.05). The RT-LAMP assay detected the ZIKV genomes of both the Asian and African lineages without cross-reacting with other arthropod-borne viruses. The sensitivity and specificity of the RT-LAMP assay were 90% (95% CI = 59.6-98.2) and 100% (95% CI = 78.5-100.0), respectively. The RT-LAMP assay detected ZIKV genome in 9 of 24 (37.5%) of the simulated clinical samples compared to 10 of 24 (41.7%) by qRT-PCR assay with a high level of concordance (κ = 0.913, P < 0.001). CONCLUSION: The RT-LAMP assay is applicable for the broad coverage detection of both the Asian and African ZIKV strains in resource-deficient settings.


Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/epidemiología , Virus Zika/clasificación , Virus Zika/genética , África/epidemiología , Asia/epidemiología , Humanos , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Infección por el Virus Zika/virología
19.
Virol Sin ; 35(6): 699-712, 2020 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33351166

RESUMEN

The on-going global pandemic of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been underway for about 11 months. Through November 20, 2020, 51 detection kits for SARS-CoV-2 nucleic acids (24 kits), antibodies (25 kits), or antigens (2 kits) have been approved by the National Medical Products Administration of China (NMPA). Convenient and reliable SARS-CoV-2 detection assays are urgently needed worldwide for strategic control of the pandemic. In this review, the detection kits approved in China are summarised and the three types of tests, namely nucleic acid, serological and antigen detection, which are available for the detection of COVID-19 are discussed in detail. The development of novel detection kits will lay the foundation for the control and prevention of the COVID-19 pandemic globally.


Asunto(s)
/diagnóstico , Juego de Reactivos para Diagnóstico , /aislamiento & purificación , /métodos , China , Técnicas de Laboratorio Clínico , Humanos , Inmunoensayo/métodos , Mediciones Luminiscentes , Técnicas de Diagnóstico Molecular/métodos , Pandemias , Reacción en Cadena en Tiempo Real de la Polimerasa
20.
PLoS One ; 15(12): e0244023, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33347478

RESUMEN

BACKGROUND: PCR is more sensitive than immunofluorescence assay (IFA) for detection of Pneumocystis jirovecii. However, PCR cannot always distinguish infection from colonization. This study aimed to compare the performance of real-time PCR and IFA for diagnosis of P. jirovecii pneumonia (PJP) in a real-world clinical setting. METHODS: A retrospective cohort study was conducted at a 1,300-bed hospital between April 2017 and December 2018. Patients whose respiratory sample (bronchoalveolar lavage or sputum) were tested by both Pneumocystis PCR and IFA were included. Diagnosis of PJP was classified based on multicomponent criteria. Sensitivity, specificity, 95% confidence intervals (CI), and Cohen's kappa coefficient were calculated. RESULTS: There were 222 eligible patients. The sensitivity and specificity of PCR was 91.9% (95% CI, 84.0%-96.7%) and 89.7% (95% CI, 83.3%-94.3%), respectively. The sensitivity and specificity of IFA was 7.0% (95% CI, 2.6%-14.6%) and 99.2% (95% CI, 95.6%-100.0%), respectively. The percent agreement between PCR and IFA was 56.7% (Cohen's kappa -0.02). Among discordant PCR-positive and IFA-negative samples, 78% were collected after PJP treatment. Clinical management would have changed in 14% of patients using diagnostic information, mainly based on PCR results. CONCLUSIONS: PCR is highly sensitive compared with IFA for detection of PJP. Combining clinical, and radiological features with PCR is useful for diagnosis of PJP, particularly when respiratory specimens cannot be promptly collected before initiation of PJP treatment.


Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Técnicas de Diagnóstico Molecular/métodos , Neumonía por Pneumocystis/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto , Anciano , Líquido del Lavado Bronquioalveolar/microbiología , Femenino , Técnica del Anticuerpo Fluorescente/normas , Humanos , Masculino , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular/normas , Pneumocystis/genética , Pneumocystis/aislamiento & purificación , Pneumocystis/patogenicidad , Neumonía por Pneumocystis/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Esputo/microbiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...