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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.
Parasite ; 28: 41, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33944774

RESUMEN

Toxoplasma gondii is an obligate intracellular protozoan parasite that causes toxoplasmosis and threatens warm-blooded animal and human health worldwide. Simple and applicable diagnostic methods are urgently needed to guide development of effective approaches for prevention of toxoplasmosis. Most molecular diagnostic tools for T. gondii infection require high technical skills, sophisticated equipment, and a controlled lab environment. In this study, we developed a loop-mediated isothermal amplification-lateral-flow-dipstick (LAMP-LFD) assay that specifically targets the 529 bp for detecting T. gondii infection. This novel portable device is universal, fast, user-friendly, and guarantees experimental sensitivity as well as low risk of aerosol contamination. Our LAMP-LFD assay has a detection limit of 1 fg of T. gondii DNA, and shows no cross-reaction with other parasitic pathogens, including Cryptosporidium parvum, Leishmania donovani, and Plasmodium vivax. We validated the developed assay by detecting T. gondii in DNA extracted from blood samples collected from 318 stray cats and dogs sampled from Deqing, Wenzhou, Yiwu, Lishui and Zhoushan cities across Zhejiang province, Eastern China. The LAMP-LFD device detected T. gondii DNA in 4.76 and 4.69% of stray cats and dogs, respectively. In conclusion, the developed LAMP-LFD assay is efficient, minimizes aerosol contamination, and is therefore suitable for detecting T. gondii across basic medical institutions and field settings.


Asunto(s)
Criptosporidiosis , Cryptosporidium , Toxoplasma , Animales , Gatos , China , Perros , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , Sensibilidad y Especificidad , Toxoplasma/genética
3.
Sci Rep ; 11(1): 9026, 2021 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-33907239

RESUMEN

The use of RT-LAMP (reverse transcriptase-loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 °C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.


Asunto(s)
/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/metabolismo , /genética , /virología , Colorimetría , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga Viral
4.
PLoS One ; 16(4): e0245423, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33852576

RESUMEN

BACKGROUND: In order to identify an inexpensive yet highly stable SARS-CoV-2 collection device as an alternative to foam swabs stored in transport media, both contrived ("surrogate") CoV-positive and patient-collected spun polyester swabs stored in dry tubes were evaluated for time- and temperature-stability using qPCR. METHODS: Surrogate specimens were prepared by combining multiple, residual SARS-CoV-2-positive clinical specimens and diluting to near-LOD levels in either porcine or human mucus ("matrix"), inoculating foam or polyester nasal swabs, and sealing in dry tubes. Swabs were then subjected to one of three temperature excursions: (1) 4°C for up to 72 hours; (2) 40°C for 12 hours, followed by 32°C for up to 60 hours; or (3) multiple freeze-thaw cycles (-20°C). The stability of extracted SARS-CoV-2 RNA for each condition was evaluated by qPCR. Separate usability studies for the dry polyester swab-based HealthPulse@home COVID-19 Specimen Collection Kit were later conducted in both adult and pediatric populations. RESULTS: Polyester swabs stored dry demonstrated equivalent performance to foam swabs for detection of low and moderate SARS-CoV-2 viral loads. Mimicking warm- and cold- climate shipment, surrogate specimens were stable following either 72 hours of a high-temperature excursion or two freeze-thaw cycles. In addition, usability studies comprised of self-collected patient specimens yielded sufficient material for molecular testing, as demonstrated by RNase P detection. CONCLUSIONS: Polyester nasal swabs stored in dry collection tubes offer a robust and inexpensive self-collection method for SARS-CoV-2 viral load testing, as viral RNA remains stable under conditions required for home collection and shipment to the laboratory.


Asunto(s)
/diagnóstico , /aislamiento & purificación , Manejo de Especímenes/métodos , Animales , Técnicas de Laboratorio Clínico/métodos , Pruebas Diagnósticas de Rutina/métodos , Humanos , Técnicas de Diagnóstico Molecular , Nasofaringe/virología , Poliésteres , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Porcinos
5.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-33806874

RESUMEN

Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs' role in several pathological conditions, from etiology to disease progression or therapy evasion, has been consolidated, including in central nervous system (CNS)-related disorders. For this review, we performed a systematic search of original works published, reporting the presence of molecular components expressed in the CNS via EVs, which have been purified from plasma, serum or cerebrospinal fluid. Our aim is to provide a list of molecular EV components that have been identified from both nonpathological conditions and the most common CNS-related disorders. We discuss the methods used to isolate and enrich EVs from specific CNS-cells and the relevance of its components in each disease context.


Asunto(s)
Biomarcadores , Enfermedades del Sistema Nervioso Central/diagnóstico , Enfermedades del Sistema Nervioso Central/metabolismo , Vesículas Extracelulares/metabolismo , Biopsia Líquida , Enfermedades del Sistema Nervioso Central/etiología , Fraccionamiento Químico/métodos , Humanos , Biopsia Líquida/métodos , Técnicas de Diagnóstico Molecular , ARN no Traducido
6.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artículo en Inglés | MEDLINE | ID: mdl-33809908

RESUMEN

Endoglin (CD105) is a type-1 integral transmembrane glycoprotein and coreceptor for transforming growth factor-ß (TGF-ß) ligands. The endoglin/TGF-ß signaling pathway regulates hemostasis, cell proliferation/migration, extracellular matrix (ECM) synthesis and angiogenesis. Angiogenesis contributes to early progression, invasion, postoperative recurrence, and metastasis in hepatocellular carcinoma (HCC), one of the most widespread malignancies globally. Endoglin is overexpressed in newly formed HCC microvessels. It increases microvessel density in cirrhotic and regenerative HCC nodules. In addition, circulating endoglin is present in HCC patients, suggesting potential for use as a diagnostic or prognostic factor. HCC angiogenesis is dynamic and endoglin expression varies by stage. TRC105 (carotuximab) is an antibody against endoglin, and three of its clinical trials were related to liver diseases. A partial response was achieved when combining TRC105 with sorafenib. Although antiangiogenic therapy still carries some risks, combination therapy with endoglin inhibitors or other targeted therapies holds promise.


Asunto(s)
Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Susceptibilidad a Enfermedades , Endoglina/genética , Endoglina/metabolismo , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Animales , Biomarcadores de Tumor , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/terapia , Endoglina/sangre , Endoglina/química , Células Endoteliales/metabolismo , Regulación Neoplásica de la Expresión Génica , Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Humanos , Neoplasias Hepáticas/patología , Técnicas de Diagnóstico Molecular , Terapia Molecular Dirigida , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Transducción de Señal , Relación Estructura-Actividad , Proteínas del Núcleo Viral/metabolismo
7.
Front Cell Infect Microbiol ; 11: 632646, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33796478

RESUMEN

A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.


Asunto(s)
/métodos , Sistemas CRISPR-Cas , ARN Viral/aislamiento & purificación , Saliva/química , Humanos , Técnicas de Diagnóstico Molecular/métodos , Pruebas en el Punto de Atención , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y Especificidad , Teléfono Inteligente , Manejo de Especímenes/métodos , Flujo de Trabajo
8.
Viruses ; 13(3)2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33807908

RESUMEN

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.


Asunto(s)
/métodos , /diagnóstico , /virología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Técnicas de Diagnóstico Molecular , Nasofaringe/virología , ARN Viral/genética , /genética , Sensibilidad y Especificidad
9.
Indian J Med Res ; 153(1 & 2): 227-232, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33818481

RESUMEN

Background & objectives: During the current COVID-19 pandemic, a large number of clinical samples were tested by real-time PCR. Pooling the clinical samples before testing can be a good cost-saving and rapid alternative for screening large populations. The aim of this study was to compare the performance characteristics, feasibility and effectiveness of pooling nasal swab and throat swab samples for screening and diagnosis of SARS-CoV-2. Methods: The pool testing was applied on a set of samples coming from low COVID-19 positivity areas. A total of 2410 samples were tested in pools of five samples each. A total of five pools of five samples each were generated and tested for E gene. Results: Of the total of 482 pools (2410 samples) 24 pools flagged positive. Later on pool de-convolution, a total of 26 samples were detected as positive for COVID-19, leading to positivity of about one per cent in the test population. For the diagnosis of individual samples, the pooling strategies resulted in cost savings of 75 per cent (5 samples per pool). Interpretation & conclusions: It was observed that testing samples for COVID-19 by reverse transcription (RT)- PCR after pooling could be a cost-effective method which would save both in manpower and cost especially for resource-poor countries and at a time when test kits were short in supply.


Asunto(s)
/métodos , Tamizaje Masivo/métodos , Análisis Costo-Beneficio , Estudios de Factibilidad , Humanos , Técnicas de Diagnóstico Molecular , Pandemias , Sensibilidad y Especificidad , Manejo de Especímenes/métodos
10.
Genes (Basel) ; 12(5)2021 04 28.
Artículo en Inglés | MEDLINE | ID: mdl-33924826

RESUMEN

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization-World Health Organization-International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region.


Asunto(s)
/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Argentina , Calibración , Humanos , Límite de Detección , /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.
Pan Afr Med J ; 38: 68, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33889234

RESUMEN

Efforts towards slowing down coronavirus (COVID-19) transmission and reducing mortality have focused on timely case detection, isolation and treatment. Availability of laboratory COVID-19 testing capacity using reverse-transcriptase polymerase chain reaction (RT-PCR) was essential for case detection. Hence, it was critical to establish and expand this capacity to test for COVID-19 in Ethiopia. To this end, using a three-phrased approach, potential public and private laboratories with RT-PCR technology were assessed, capacitated with trained human resource and equipped as required. These laboratories were verified to conduct COVID-19 testing with quality assurance checks regularly conducted. Within a 10-month period, COVID-19 testing laboratories increased from zero to 65 in all Regional States with the capacity to conduct 18,454 tests per day. The success of this rapid countrywide expansion of laboratory testing capacity for COVID-19 depended on some key operational implications: the strong laboratory coordination network within the country, the use of non-virologic laboratories, investment in capacity building, digitalization of the data for better information management and establishing quality assurance checks. A weak supply chain for laboratory reagents and consumables, differences in the brands of COVID-19 test kits, frequent breakdowns of the PCR machines and inadequate number of laboratory personnel following the adaption of a 24/7 work schedule were some of the challenges experienced during the process of laboratory expansion. Overall, we learn that multisectoral involvement of laboratories from non-health sectors, an effective supply chain system with an insight into the promotion of local production of laboratory supplies were critical during the laboratory expansion for COVID-19 testing. The consistent support from WHO and other implementing partners to Member States is needed in building the capacity of laboratories across different diagnostic capabilities in line with International Health Regulations. This will enable efficient adaptation to respond to future public health emergencies.


Asunto(s)
/métodos , Laboratorios/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , /normas , Creación de Capacidad , Equipos y Suministros/estadística & datos numéricos , Etiopía , Humanos , Laboratorios/estadística & datos numéricos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Garantía de la Calidad de Atención de Salud , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas
13.
Int J Mol Sci ; 22(6)2021 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-33808647

RESUMEN

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies worldwide. HCC is associated with several etiological factors, including HBV/HCV infections, cirrhosis, and fatty liver diseases. However, the molecular mechanism underlying HCC development remains largely elusive. The advent of high-throughput sequencing has unveiled an unprecedented discovery of a plethora of long noncoding RNAs (lncRNAs). Despite the lack of coding capacity, lncRNAs have key roles in gene regulation through interacting with various biomolecules. It is increasingly evident that the dysregulation of lncRNAs is inextricably linked to HCC cancer phenotypes, suggesting that lncRNAs are potential prognostic markers and therapeutic targets. In light of the emerging research in the study of the regulatory roles of lncRNAs in HCC, we discuss the association of lncRNAs with HCC. We link the biological processes influenced by lncRNAs to cancer hallmarks in HCC and describe the associated functional mechanisms. This review sheds light on future research directions, including the potential therapeutic applications of lncRNAs.


Asunto(s)
Biomarcadores de Tumor , Carcinoma Hepatocelular/genética , Susceptibilidad a Enfermedades , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/genética , ARN Largo no Codificante , Animales , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Transformación Celular Neoplásica/genética , Manejo de la Enfermedad , Genes Supresores de Tumor , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Técnicas de Diagnóstico Molecular , Terapia Molecular Dirigida , Oncogenes , Transducción de Señal/efectos de los fármacos
14.
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
15.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-33671091

RESUMEN

Although molecular testing, and RT-qPCR in particular, has been an indispensable component in the scientific armoury targeting SARS-CoV-2, there are numerous falsehoods, misconceptions, assumptions and exaggerated expectations with regards to capability, performance and usefulness of the technology. It is essential that the true strengths and limitations, although publicised for at least twenty years, are restated in the context of the current COVID-19 epidemic. The main objective of this commentary is to address and help stop the unfounded and debilitating speculation surrounding its use.


Asunto(s)
/métodos , /virología , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , /aislamiento & purificación , Técnicas de Laboratorio Clínico/métodos , Humanos , ARN Viral/análisis , ARN Viral/genética , Sensibilidad y Especificidad
16.
Euro Surveill ; 26(10)2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33706863

RESUMEN

BackgroundSensitive molecular diagnostics and correct test interpretation are crucial for accurate COVID-19 diagnosis and thereby essential for good clinical practice. Furthermore, they are a key factor in outbreak control where active case finding in combination with isolation and contact tracing are crucial.AimWith the objective to inform the public health and laboratory responses to the pandemic, we reviewed current published knowledge on the kinetics of SARS-CoV-2 infection as assessed by RNA molecular detection in a wide range of clinical samples.MethodsWe performed an extensive search on studies published between 1 December 2019 and 15 May 2020, reporting on molecular detection and/or isolation of SARS-CoV-2 in any human laboratory specimen.ResultsWe compiled a dataset of 264 studies including 32,515 COVID-19 cases, and additionally aggregated data points (n = 2,777) from sampling of 217 adults with known infection timeline. We summarised data on SARS-CoV-2 detection in the respiratory and gastrointestinal tract, blood, oral fluid, tears, cerebrospinal fluid, peritoneal fluid, semen, vaginal fluid; where provided, we also summarised specific observations on SARS-CoV-2 detection in pregnancy, infancy, children, adolescents and immunocompromised individuals.ConclusionOptimal SARS-CoV-2 molecular testing relies on choosing the most appropriate sample type, collected with adequate sampling technique, and with the infection timeline in mind. We outlined knowledge gaps and directions for future well-documented systematic studies.


Asunto(s)
/diagnóstico , Técnicas de Diagnóstico Molecular , Humanos , Sensibilidad y Especificidad
17.
Elife ; 102021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33779548

RESUMEN

Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. The test has two steps: (1) heat saliva with a stabilization solution and (2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections.


Asunto(s)
/diagnóstico , Portador Sano/diagnóstico , Portador Sano/virología , Saliva/virología , /metabolismo , Humanos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/genética , Sensibilidad y Especificidad , Manejo de Especímenes/métodos
18.
Int J Mol Sci ; 22(5)2021 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-33669073

RESUMEN

African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this "easy lab" workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the "easy lab" strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the "easy lab" concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the "easy lab" strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.


Asunto(s)
Virus de la Fiebre Porcina Africana/aislamiento & purificación , Fiebre Porcina Africana/diagnóstico , ADN Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sus scrofa/virología , Porcinos/virología , Fiebre Porcina Africana/sangre , Fiebre Porcina Africana/epidemiología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Animales , ADN Viral/aislamiento & purificación , Brotes de Enfermedades/veterinaria , Alemania , Estándares de Referencia , Sensibilidad y Especificidad
19.
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
20.
J Dairy Sci ; 104(5): 5152-5165, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33663822

RESUMEN

Due to the lack of specific genes for rapid detection methods of Cronobacter sakazakii in food samples, whole genome sequence analysis was performed in this investigation using the basic local alignment search tool. Forty-two DNA fragments unique to C. sakazakii were mined, then primers were designed and screened by PCR and loop-mediated isothermal amplification (LAMP). Two primer sets, CS1 and CS31, were found as specific and stable primers, with their corresponding nucleic acid targets the CSK29544_00235 gene and CSK29544_03484 gene, respectively. Furthermore, compared with 3 genes reported previously, these 2 genes were verified as more specific to C. sakazakii among Cronobacter species, by sequence similarity alignment using Cronobacter MLST databases (http://pubmlst.org/cronobacter). The specificity of the LAMP reaction approached 100% by using 48 bacterial strains, which included 22 C. sakazakii strains. Subsequently, LAMP was combined with visual lateral flow dipstick (LFD) based on the above 2 nucleic acid targets, and was demonstrated as a rapid, efficient method with high specificity. Finally, the detection sensitivity of this assay system for pure cultures and artificially contaminated milk was measured as 4.5 × 100 cfu/mL and 5.7 × 101 cfu/g, respectively. Total time to detection for this assay was within 2 h. Thus, the establishment of this LAMP-LFD method shows great significance and potential for rapid detection of C. sakazakii in powdered infant formula.


Asunto(s)
Cronobacter sakazakii , Cronobacter , Ácidos Nucleicos , Animales , Cronobacter/genética , Cronobacter sakazakii/genética , Microbiología de Alimentos , Fórmulas Infantiles , Técnicas de Diagnóstico Molecular , Tipificación de Secuencias Multilocus/veterinaria , Técnicas de Amplificación de Ácido Nucleico , Polvos
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