Persona: Cabria Ramos, Juan Carlos
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Cabria Ramos
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Juan Carlos
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Publicación Restringido Rapid identification of Bacillus anthracis by real-time PCR with dual hybridization probes in environmental swabs(Elsevier, 2017-11-04) Bassy Alvarez, Olga; Jiménez, Oscar; Ortega García, María Victoria; Granja, Carmen; Cabria Ramos, Juan CarlosIn the present study, we report the development of a real-time PCR assay for the identification of Bacillus anthracis, based on the amplification of a unique chromosomal marker, the E4 sequence, with dual hybridization probes. The assay was evaluated using a panel of ten B. anthracis strains, two B. anthracis isolates from human clinical samples, 12 B. anthracis environmental swabs and 40 non- B. anthracis strains. All 12 B. anthracis strains and clinical isolates were correctly detected, and the method did not show cross-reactions with other micro-organisms. Likewise, the E4 sequence was not found in those strains of B. thuringiensis and B. cereus closely related (homology > 90%) to B. anthracis by computer analysis. On the other hand, this molecular assay showed a high analytical sensitivity, 3.5 genome equivalents per reaction at 95% probability. Furthermore, the real-time PCR assay allowed sequence-specific detection of the amplicon (melting peak with a Tm of 63.5 °C ± 0.5 °C) without post-amplification procedures, which offers an additional advantage over other qPCR assays for B. anthracis detection. Finally, the performance of the method was successfully evaluated in 12 environmental samples. In summary, we have developed a rapid and specific method for the molecular identification of Bacillus anthracis in environmental samples.Publicación Acceso Abierto Diseño de oligonucleótidos sonda para la detección de virus de interés en biodefensa(Sanidad Militar, 2018) González López, Laura; Peraile, Inés; Rozas Sanz, Gabriel; Cabria Ramos, Juan Carlos; Lorenzo Lozano, PalomaAntecedentes: Los virus causan enfermedades en el hombre como la gripe, la rabia, la fiebre amarilla o la fiebre hemorrágica. Además, presentan características como alta variabilidad genética, virulencia y fácil transmisión y producción con infraestructuras mínimas, lo que les convierte en un problema de salud pública y de bioseguridad. Por todo ello, desarrollar sistemas de biodetección precisos y versátiles es un reto ante el cual, la tecnología de micromatrices de ADN, se presenta como un sistema de ideal. Objetivos: Diseño de oligonucleótidos sonda para la detección de especies pertenecientes a nueve familias de virus de interés en biodefensa. Material y Métodos: Se seleccionaron familias de virus de interés en biodefensa, se buscaron los genomas completos de los virus de referencia de cada una de ellas en las bases de datos (GenBank) y se identificaron fragmentos de 60 nucleótidos que cumplieran determinados condicionantes estructurales, evaluándose con BLASTN su capacidad de hibridación cruzada. Resultados: Se obtuvieron los genomas de referencia de virus pertenecientes a nueve familias. Se diseñaron un total de 54 nucleótidos sonda, seis de ellos correspondientes al virus de la gripe A tipo H1N1 y se clasificaron las secuencias según su índice de identidad, permitiendo predecir la capacidad diagnóstica de las sondas diseñadas. Conclusiones: Se han encontrado secuencias suficientes para identificar nueve familias de virus, de interés en la biodefensa, mediante la tecnología de hibridación de micromatrices de ADN.Publicación Restringido Reuse of nanofibers in a Biological Warfare Agent detection nanophotonic sensing device(CBRNE Research and Inovation, 2019-05-29) Peraile, Inés; Lorenzo Lozano, Paloma; González López, Laura; Murillo, Nieves; Dabbagh Escalante, Nushin Alba; Rozas Sanz, Gabriel; Cabria Ramos, Juan Carlos; Gil García, Matilde"The use of biological injurious agent ravages not only at human population but also at livestock and crops [1], causing effects both on human health and on the economy [2]. Therefore, the fast and specific detection of these biological injurious agents is necessary in many fields such as environmental control, clinic diagnostic, food industry, Security and Defence. Therefore, much effort has been devoted to the development of new technologies for their detection and identification. The priority objective is to carry out a low cost, easy-of-use and specific device to be used on site and able to be miniaturized [3]. In order to meet this need, the specificity of the antigen-antibody binding is applied in a multitude of biosensors. Among these immuno-biosensors, biological warfare agent detection equipment stands out. Provided both the high costs for this equipment and the need for the first responders to move into the CBRN accident area, it could be desirable to have reusable detection devices. In the OPTONANOSENS project, an immuno-biosensor with nanophotonic sensing has been developing. In this device, nylon nanofibers with 5% pyridine, manufactured by Tecnalia laid on a metallic support is used to immobilized antibodies [4,5]. The aims of this work are both to make a comparison between several procedures which let these nanofibers to be reused in different detection events and the optimisation of the more suitable procedure which allows a repetitive use and reliable of the biofunctionalized nanofibers in the final sensing device."Publicación Restringido lnactivation and identification of Bacillus anthracis spores(Wageningen Academic Publishers, 2013-12) Gil García, Matilde; Peraile, Inés; Jiménez Pérez, M. V.; Cabria Ramos, Juan Carlos; Lorenzo Lozano, PalomaBacillus anthracis endospores must be handled ata biosafety level-3 (BSL-3} eontainment facility, therefore an appropriate inaetivation procedure is essential in order to both detect thcm in a reliable and rapid way and to avoid human infections from antrax-contamined samples if a biosafety level-3 laboratory is not availablc. Unfortunatcly, donnant spores exhibit incredible hardiness against gennicidal agents. The most effective methods for inactivating spores are usually the chemical treatment, heat inactivation (boiling, moist hcat, dry heat) and irTadiation (microwave, UV, gamma, electron beam). The inactivation method used must alter thc cell structure as little as possible in order to ensure a corree! diagnosis by diferent techniques. Several kinds ofphysical and chemical inactivation methods wcrc assessed in order to define the conditions that ensure inactivation of endospores of B. anthracis 34F2 Sterné and pcrmit, at the same time, its identification by PCR and immunoassays techniques.Publicación Restringido Development of a specific real-time PCR assay for simultaneous detection and differentiation of Coxiella burnetii strains from environmental soil samples(Oxford University Press, 2023-02-25) Fernández Carrillo, Juan; Del Olmo Monge, Javier; Sellek Cano, Ricela, Ricela; Ortega García, María Victoria; Cabria Ramos, Juan Carlos; Bassy Alvarez, OlgaCoxiella burnetii, the causative agent of Q fever, is a small, coccoid, Gram-negative strict intracellular pathogen. One of the most common ways of acquiring Q fever is through inhalation of aerosols containing the bacteria. Because C. burnetii is highly infectious, spreads easily through the air, and is very resistant to environmental conditions, it is considered a biological threat. This paper presents the development and validation of a specific real-time polymerase chain reaction (real-time PCR or qPCR) assay for the detection of C. burnetii, based on the amplification of a fragment of the isocitrate dehydrogenase (icd) encoding gene. This real-time PCR is highly specific, reproducible, and sensitive, allowing the detection of as few as 5 genome equivalents (GEs) of C. burnetii per reaction. The method enables a rapid preliminary differentiation among strains, based on a point mutation at nucleotide 745 of the icd gene. The assay was successfully evaluated in environmental soil samples; a limit of detection of 3 × 104 colony forming units per 0.5 g of soil (∼3 GEs per reaction) was achieved. The newly developed real-time PCR offers a valuable tool for differential detection of C. burnetii strains in environmental soil samples.Publicación Acceso Abierto Study of the reusability and stability of nylon nanofibres as an antibody immobilisation surface(Beilsein Institut, 2024-01-15) Peraile, Inés; Gil García, Matilde; González López, Laura; Dabbagh Escalante, Nushin Alba; Cabria Ramos, Juan Carlos; Lorenzo Lozano, Paloma; Ministerio de Economía y Competitividad (MINECO)In the case of a biological threat, early, rapid, and specific detection is critical. In addition, ease of handling, use in the field, and low-cost production are important considerations. Immunological devices are able to respond to these needs. In the design of these immunological devices, surface antibody immobilisation is crucial. Nylon nanofibres have been described as a very good option because they allow for an increase in the surface-to-volume ratio, leading to an increase in immunocapture efficiency. In this paper, we want to deepen the study of other key points, such as the reuse and stability of these nanofibres, in order to assess their profitability. On the one hand, the reusability of nanofibres has been studied using different stripping treatments at different pH values on the nylon nanofibres with well-oriented antibodies anchored by protein A/G. Our study shows that stripping with glycine buffer pH 2.5 allows the nanofibres to be reused as long as protein A/G has been previously anchored, leaving both nanofibre and protein A/G unchanged. On the other hand, we investigated the stability of the nylon nanofibres. To achieve this, we analysed any loss of immunocapture ability of well-oriented antibodies anchored both to the nylon nanofibres and to a specialised surface with high protein binding capacity. The nanofibre immunocapture system maintained an unchanged immunocapture ability for a longer time than the specialised planar surface. In conclusion, nylon nanofibres seem to be a very good choice as an antibody immobilisation surface, offering not only higher immunocapture efficiency, but also more cost efficiency as they are reusable and stable.Publicación Acceso Abierto Biofuncionalización de superficies de nylon con anticuerpos antiricina para su uso en inmunobiosensores(Ministerio de Defensa, 2019-02-15) Peraile, Inés; Gil García, Matilde; Murillo, Nieves; Maudes, Jon; Rozas Sanz, Gabriel; González López, Laura; Dabbagh Escalante, Nushin Alba; Pérez, Ana; Cabria Ramos, Juan Carlos; Lorenzo Lozano, Paloma"Los agentes de guerra biológica constituyen un riesgo creciente en la actualidad. Por ello, la mayoría de los países invierten en la investigación y el desarrollo de técnicas de detección e identificación de dichos agentes, siendo un objetivo prioritario el desarrollo de dispositivos que permitan una identificación temprana in situ lo más específica y sensible posible. Dentro de estos dispositivos, los inmunobiosensores (basados en técnicas inmunológicas) resultan ser los candidatos de elección, puesto que la unión antígeno-anticuerpo es rápida y altamente específica. En un inmunobiosensor el tipo de sensado determinará el umbral de detección, mientras que la especificidad y sensibilidad de la detección dependerá de los anticuerpos seleccionados y de la forma en que estos son inmovilizados. Es por ello por lo que, tanto la superficie como el método de inmovilización, determinarán la densidad y orientación del anticuerpo inmovilizado y, en consecuencia, la eficiencia de la unión antígeno-anticuerpo y el éxito del sistema de inmunocaptura. La elección de un modelo experimental de inmunocaptura similar al modelo final en el inmunobiosensor es fundamental. Así, se diseñó un modelo de inmunocaptura en el que se marcaron con diferentes fluoróforos el anticuerpo y el antígeno, y se determinaron diferentes parámetros (densidad de anticuerpo inmovilizado, de antígeno inmunocapturado y eficacia del sistema) con el fin de realizar el seguimiento del proceso de inmovilización y de inmunocaptura. Dicho modelo de inmunocaptura se empleó para el estudio y la comparación de diferentes superficies y métodos de inmovilización. En una primera fase, estos estudios se desarrollaron con simulantes de agentes de guerra biológicos. En una segunda fase, se emplearon agentes reales, como ricina. Nuestro modelo de inmunocaptura y los parámetros considerados han resultado ser adecuados y versátiles para el estudio y comparación de diferentes superficies y modelos de inmovilización."Publicación Restringido Biofunctionalization of nylon nanofibers to be used in immunobiosensor for biological warfare agents detecting(Formatex Research Center, 2018) Peraile, Inés; Lorenzo Lozano, Paloma; Murillo, N.; Maudes, J.; Rozas Sanz, Gabriel; Pérez Márquez, Ana; González López, Laura; Cabria Ramos, Juan Carlos; Gil García, MatildeThe use of biological warfare agents involves a growing threat to society. Thus, the most countries have been forced to increase resources in research for their detection and identification. One of the critical points in the effective fight against these agents is the development of devices that allow their detection and early identification. The best choice is the immunobiosensors easy to use on-site. However, how the antibody is attached to the biosensor surface, in terms of density and orientation, will determine the diagnosis capability of the device. In this study, both a functional nanofiber able to increase the surface / volume relation, and a chemicallysimilar planar membrane were used as support for the immobilization of antibodies. Different antibody immobilization systems were carried out to biofunctionalize both surfaces: passive adsorption, covalent bond by glutaraldehyde and well-oriented immobilization by protein A/G. Our results showed that nanofibers in combination with protein A/G were a very effective immunocapture system for being used in a biosensor.Publicación Acceso Abierto Spanish Outbreak Isolates Bridge Phylogenies of European and American Bacillus anthracis(MDPI - Multidisciplinary Digital Publishing Institute, 2023-03-29) Bassy Alvarez, Olga; Antwerpen, Markus; Ortega García, María Victoria; Ortega Sánchez, María Jesús; Bouzada, José Antonio; Cabria Ramos, Juan Carlos; Grass, Gregor; Ortega García, María VictoriaThe geographical origin of a major present-day phylogenetic group (A branch WNA; A.Br.WNA) of American Bacillus anthracis is controversial. One hypothesis postulated that the anthrax pathogen reached North America via a then-existing land bridge from northeastern Asia thousands of years ago. A competing hypothesis suggested that B. anthracis was introduced to America a couple of hundred years ago, related to European colonization. The latter view is strongly supported by genomic analysis of a group of French B. anthracis isolates that are phylogenetically closely related to the North American strains of the A branch A.Br.WNA clade. In addition, three West African strains also belong to this relationship group. Recently, we have added a Spanish strain to these close relatives of the WNA lineage of American B. anthracis. Nevertheless, the diversity of Spanish B. anthracis remains largely unexplored, and phylogenetic links to European or American relatives are not well resolved. Here, we genome sequenced and characterized 29 new B. anthracis isolates (yielding 18 unique genotypes) from outbreaks in west central and central Spain in 2021. Applying comparative chromosomal analysis, we placed the chromosomes of these isolates within the established phylogeny of the A.Br.008/009 (A.Br.TEA) canonical SNP group. From this analysis, a new sub-clade, named A.Br.11/ESPc, emerged that constitutes a sister group of American A.Br.WNA.Publicación Acceso Abierto Evaluación de detectores de aerosoles biológicos en túnel de viento(Ministerio de Defensa, 2019-02-15) Dabbagh Escalante, Nushin Alba; Rozas Sánz, Gabriel; Peraile, Inés; González López, Laura; Cabria Ramos, Juan Carlos; Lorenzo Lozano, Paloma"Durante el siglo pasado, los avances en biotecnología y bioquímica han simplificado el desarrollo y la producción de armas biológicas. Estas pueden ser más potentes que las armas convencionales o las armas químicas por su alta contagiosidad, fácil difusión y la cantidad mínima de organismos requeridos para causar enfermedad y/o muerte. Los agentes biológicos que tienen mayor probabilidad de ser utilizados como armas biológicas son bacterias, rickettsias, virus y toxinas, y la vía de dispersión de mayor interés desde el punto de vista de la seguridad y la defensa son los bioaerosoles. Desde los ataques con ántrax producidos en 2001 en EE.UU., los países han incrementado considerablemente los esfuerzos en investigación para el desarrollo de nuevas tecnologías que permitan una detección temprana de las amenazas biológicas. Disponer de sistemas de detección en tiempo real y con una elevada sensibilidad permite minimizar los riesgos de exposición a este tipo de agentes, ya que la información obtenida puede ser utilizada para adoptar medidas de protección y prevención apropiadas a la amenaza detectada. El área de defensa biológica del Departamento de Sistemas de Defensa NBQ y Materiales Energéticos de la Subdirección General de Sistemas Terrestres del INTA (Campus La Marañosa) ha puesto a punto una metodología para evaluar nuevos equipos de detección de aerosoles biológicos. La eficacia de estos equipos se ha analizado en términos de tiempo de respuesta, sensibilidad y capacidad para diferenciar entre eventos biológicos y no biológicos. Los ensayos se han realizado en un túnel de viento (diseñado por la empresa Ibatech) sobre el que se inyectan aerosoles biológicos de concentraciones conocidas. Para la generación de la nube se han utilizado simulantes de agentes de guerra biológica (Bacillus thuringiensis como simulante de Bacillus anthracis y Pantoea agglomerans como simulante de Yersinia pestis)."
