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Sci Rep ; 11(1): 16430, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385527


Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.

Teste para COVID-19/métodos , COVID-19/virologia , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/isolamento & purificação , COVID-19/diagnóstico , Humanos , Pandemias , Portugal/epidemiologia , RNA Viral/genética , SARS-CoV-2/genética , Saliva/química , Saliva/virologia , Sensibilidade e Especificidade
ChemistryOpen ; 6(4): 562-570, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28794952


We report the development of new chemical probes for cereulide, a toxic metabolite produced by specific strains of Bacillus cereus, through displacement of potassium cations from a preformed specific complex and a subsequent change in the fluorescence emission. For this purpose, we designed fluorescent probes for potassium cations that were suitable for displacement assays with cereulide from organic extracts. The fluorescence detection of natural cereulide in rice samples was achieved by using synthetic cereulide as a reference and a potassium fluorescent reporter, and this was found to be useful as a portable and fast method for the in situ detection of cereulide in food extracts. To study the fate of cereulide in live cells, we designed a procedure that was suitable for live-cell microscopy imaging of HeLa cells by comparing the cellular location of the potassium fluorogenic probe, which stained intracellular endolysosomes, in the absence and presence of cereulide; we concluded that in the presence of cereulide, the fluorescence of the probe was decreased because of complexation of the potassium ions by cereulide.

Int J Paleopathol ; 16: 5-13, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28290311


Calcification, or mineralisation, can occur as part of a natural process, or by pathological processes. The purpose of this work is to examine an unidentified semi-spherical and perforate hollow mass, found near the pelvis of an adult female, dated 12th-13th century AD, exhumed of the Church of Santo Domingo de Silos (Prádena del Ricón, Madrid, Spain). The mass was examined by SEM and Energy Dispersive X-Ray Spectroscopy. These procedures revealed a heterogeneous inner surface with both smooth and irregular areas. A larger spherical and several smaller crescent-shaped perforations were noticed. X-ray microanalysis revealed the presence of the elements C, K, P, Ca, Al, Si, Fe, and Mg. The co-localisation of Ca and P suggests that they may be combined in a mineral matrix, likely formed in vivo. Other minerals probably came from the soil, although Fe could be related to the presence of blood. The macroscopic and microscopic appearances, chemical composition, and location of the calcified mass point to a possible hydatid cyst from Echinococcus granulosus, common in agricultural populations. This study used a suite of analytical techniques that are useful in the diagnosis of unknown calcified masses and can, therefore, be recommended for use in future analytical work.

Equinococose/história , Equinococose/patologia , Adulto , Animais , Calcinose/patologia , Echinococcus granulosus , Feminino , História Medieval , Humanos , Paleontologia , Espanha