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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 Carlos
In 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.
Restringido
Recovery of Francisella tularensis from soil samples by filtration and detection by real-time PCR and cELISA
(Royal Society of Chemistry, 2008-01-17) Sellek Cano, Ricela; Jiménez, Oscar; Aizpurua, Carmen; Fernández Frutos, Begoña; De León, Patricia; Camacho, Maite; Fernández Moreira, Daniel; Ybarra, Carmen; Cabria Ramos, Juan Carlos
The aim of this study was to develop a specific and highly sensitive method able to detect very low concentrations of Francisella tularensis in soil samples by real-time PCR (qPCR) with SYBR Green I. tul4gene, which encodes the 17-kDa protein (TUL4) in F. tularensis strains, was amplified using a LightCycler (LC) device. We achieved a detection limit of 0.69 fg of genomic DNA from F. tularensis subp. holarctica live vaccine strain (LVS), corresponding to a value less than 3.4 genome equivalents per reaction. The qPCR was shown to be specific, highly sensitive and reproducible. In addition, we evaluated 2 new methods for recovering bacteria from soil based on 1-step filtration using glass fiber filters and PVDF filters. These filtration methods enabled us to recover F. tularensis efficiently from soil samples. As few as 50 CFU per 0.5 g of soil were detected by qPCR. Capture enzyme-linked immunosorbent assay (cELISA) allowed us to detect and quantify the amount of bacteria recovered from soil by an immunological method. Although qPCR was more sensitive than cELISA, we did not observe substantial differences in the amount of bacteria quantified by both methods.
Restringido
Preliminary validation of real-time PCR assays for the identification of Yersinia pestis
(De Gruyter Brill, 2008-07-21) Tomaso, Herbert; Jacob, Daniela; Eickhoff, Meike; Scholz, Holger; Al Dahouk, Sascha; Kattar, Mireille; Reischl, Udo; Plicka, Helga; Strand Olsen, Jaran; Nikkari, Simo; Matero, Pirjo; Beuret, Christian; Ciammaruconi, Andrea; Lista, Florigio; Gala, Jean Luc; Broll, Hermann; Appel, Bernd; Sellek Cano, Ricela; Ybarra, María del Carmen; Broekhuijsen, Martien; Indra, Alexander; Petersen, Roger; Neubauer, Heinrich
Background: Yersinia pestis (Y. pestis) is a zoonotic bacterium mainly circulating among rodents and their fleas. Transmission to humans can cause bubonic, pneumonic or septicemic plague with a high case-fatality rate. Therefore, rapid and reliable diagnostic tools are crucial. The objective of this study was to assess the inter-laboratory reproducibility of in-house developed real-time PCR assays for the identification of Y. pestis. Methods: A total of four samples of quantified Y. pestis DNA and two blank samples were sent blinded to 14 laboratories. To standardize the procedures, oligonucleotides were provided and the same instrument platform and a commercial mastermix were used. The participants were requested to report their results including cycle threshold and melting temperature values. Results: All participating laboratories were able to perform the real-time PCR assays according to the protocols provided and identified the samples containing Y. pestis DNA correctly. Significant differences between the reference laboratory and participating laboratories were observed in cycle threshold values and melting temperatures. This, however, did not adversely affect the interpretation of results. Conclusions: Our real-time PCR system proved to be highly reproducible and has the potential of complementing the diagnostic tools for rapid identification of Y. pestis isolates. Further steps of validation are needed to determine diagnostic accuracy and predictive values with clinical samples.
Acceso Abierto
Phenotypic and genetic analyses of 111 clinical and environmental O1, O139, and non-O1/O139 Vibrio cholerae strains from different geographical areas
(Cambridge University Press, 2012-08-05) Sellek Cano, Ricela; Niemcewicz, Marcin; Olsen, Jaran; Bassy Alvarez, Olga; Lorenzo Lozano, P.; Martí, L.; Roszowiak, A.; Kocik, J.; Cabria Ramos, Juan Carlos
A total of 111 clinical and environmental O1, O139 and non-O1/O139 Vibrio cholerae strains isolated between 1978 and 2008 from different geographical areas were typed using a combination of methods: antibiotic susceptibility, biochemical test, serogroup, serotype, biotype, sequences containing variable numbers of tandem repeats (VNTRs) and virulence genes ctxA and tcpA amplification. As a result of the performed typing work, the strains were organized into four clusters: cluster A1 included clinical O1 Ogawa and O139 serogroup strains (ctxA + and tcpA +); cluster A2 included clinical non-O1/O139 strains (ctxA − and tcpA −), as well as environmental O1 Inaba and non-O1/O139 strains (ctxA − and tcpA −/tcpA +); cluster B1 contained two clinical O1 strains and environmental non-O1/O139 strains (ctxA − and tcpA +/tcpA −); cluster B2 contained clinical O1 Inaba and Ogawa strains (ctxA + and tcpA +). The results of this work illustrate the advantage of combining several typing methods to discriminate between clinical and environmental V. cholerae strains.
Acceso Abierto
OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay
(Springer Nature Link, 2014-06-18) Paauw, Armand; Trip, Hein; Niemcewicz, Marcin; Sellek Cano, Ricela; Heng, Jonathan; Mars Groenendijk, Roos; De Jong, Ad; Majchrzykiewicz Koehorst, Joanna; Olsen, Jaran; Tsivtsivadze, Evgeni
Background Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks. Results The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay. Conclusions The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved.
Acceso Abierto
Diseño de oligonucleótidos sonda para la detección de virus de interés en biodefensa
(Sanidad Militar, 2018) González López, L.; Peraile, Inés; Rozas Sanz, Gabriel; Cabria Ramos, Juan Carlos; Lorenzo Lozano, P.
Antecedentes: 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.
Acceso Abierto
Cocaine potentiates MDMA-induced oxidative stress but not dopaminergic neurotoxicity in mice: implications for the pathogenesis of free radical-induced neurodegenerative disorders
(Springer Nature, 2013-05-17) Peraile, Inés; Granado, Noelia; Torres, Elisa; Gutiérrez López, María Dolores; Moratalla, Rosario; Colado, Isabel; O’Shea, Esther; Ministerio de Ciencia e Innovación (MICINN); Comunidad de Madrid; Ministerio de Sanidad y Política Social; Instituto de Salud Carlos III (ISCIII); Universidad Complutense de Madrid (UCM)
The drugs of abuse 3,4-methylenedioxymethamphetamine (MDMA; “ecstasy”) and cocaine both increase the generation of free radicals, and in the case of MDMA, this increase in oxidative stress is involved in the dopaminergic neurotoxicity produced by the drug in mice. Oxidative stress processes are also involved in the pathogenesis of several neurodegenerative diseases.
Acceso Abierto
Protocolo para la identificación rápida y sensible de ricina en muestras ambientales ante una alerta biológica
(Sanidad Militar, 2017) Peraile, Inés; Lorenzo Lozano, P.; Gil García, M.; González López, L.; Cabria Ramos, Juan Carlos; Jiménez Pérez, M. V.
Introducción: La ricina es una toxina muy potente que ha adquirido importancia por su potencial uso como arma biológica, fundamentalmente en forma pulverulenta. El desarrollo de métodos que permitan una detección rápida y temprana tras la exposición a la toxina permitiría reducir las tasas de morbilidad y mortalidad asociadas. Ante una alerta/amenaza biológica con una muestra sospechosa de contener ricina, la Red de Laboratorios de Alerta Biológica (RELAB), infraestructura de naturaleza científico-técnica creada mediante la Orden PRE/305/2009, autoriza el envío de la muestra al Instituto Nacional de Técnica Aerospacial (INTA), uno de los laboratorios de referencia que integran esta Red. Objetivos: Desarrollo de un protocolo para la detección de ricina basado en una doble detección, un ensayo inmunológico y un análisis proteico, para aumentar la fiabilidad del diagnóstico además de acortar sensiblemente el tiempo de respuesta del laboratorio. Material y Métodos: El diagnóstico inmunológico con anticuerpos in house combinados en un ELISA tipo Sandwich. El diagnóstico proteico mediante la técnica SDS-PAGE (electroforesis en gel de poliacrilamida con dodecilsulfato sódico) para determinar el tamaño y la estructura de las distintas proteínas de la muestra. Resultados: La optimización del marcado de los anticuerpos de detección así como la preparación y almacenamiento de placas previamente tapizadas/bloqueadas permite conseguir un ensayo muy sensible (<1 ng/mL) en un tiempo inferior a cuatro horas.
Acceso Abierto
Optimización del proceso de inmovilización de anticuerpos en inmunobiosensores
(Sanidad Militar, 2018) Peraile, Inés; Gil García, M.; Guamán Collaguazo, C. E.; González López, L.; Cabria Ramos, Juan Carlos; Lorenzo Lozano, P.
Antecedentes: La detección rápida y específica de agresivos biológicos es fundamental en diversos campos como control ambiental, diagnóstico clínico, industria alimentaria, seguridad y defensa. La especificidad de la unión antígeno-anticuerpo es empleada en multitud de biosensores, como equipos de identificación de agentes de guerra biológicos, pero el cómo se una ese anticuerpo en la superficie del biosensor, en términos de densidad, orientación, y estabilidad determinará la capacidad diagnóstica del dispositivo. Objetivo: Desarrollo de procesos de inmovilización de anticuerpos en superficies planares que permitan una unión antígeno-anticuerpo eficiente, para su posterior uso en dispositivos inmunológicos de sensado. Material y Métodos: Se ensayaron tres métodos de inmovilización del anticuerpo-fluoresceína sobre una membrana Zprobe: adsorción pasiva, unión covalente con glutaraldehído (0,5 %) y unión orientada con proteína mediadora A/G (5 y 10 µg). Se seleccionó albúmina sérica bovina-ficoeritrina como simulante de toxina proteica. Resultados: El porcentaje de retención del anticuerpo inmovilizado durante el proceso de inmunocaptura fue similar en los métodos ensayados. La densidad del anticuerpo inmovilizado fue mayor en la inmovilización con glutaraldehído y menor con proteína A/G. Sin embargo, respecto a la eficiencia de la inmunocaptura del antígeno, la inmovilización del anticuerpo con glutaraldehído fue la menos eficiente frente a la inmovilización con proteínas A/G, que resultó ser la más eficaz. Conclusiones: La utilización de glutaraldehído en la inmovilización del anticuerpo, aunque incrementa la densidad de unión del mismo sobre una membrana Zprobe, interfiere en el proceso de inmunodetección antigénica, mientras que el uso de la proteína mediadora A/G permiten un sistema de inmunocaptura más eficiente, con una menor densidad de anticuerpo inmovilizado.
Acceso Abierto
Passive exercise adaptation for ankle rehabilitation based on learning control framework
(MDPI AG, 2020-09-01) Abu Dakka, F.J.; Valera, A.; Escalera, J.A.; Abderrahim, M.; Page, A.; Mata, V.; Ministerio de Economía y Competitividad (MINECO)
Ankle injuries are among the most common injuries in sport and daily life. However, for their recovery, it is important for patients to perform rehabilitation exercises. These exercises are usually done with a therapist’s guidance to help strengthen the patient’s ankle joint and restore its range of motion. However, in order to share the load with therapists so that they can offer assistance to more patients, and to provide an efficient and safe way for patients to perform ankle rehabilitation exercises, we propose a framework that integrates learning techniques with a 3-PRS parallel robot, acting together as an ankle rehabilitation device. In this paper, we propose to use passive rehabilitation exercises for dorsiflexion/plantar flexion and inversion/eversion ankle movements. The therapist is needed in the first stage to design the exercise with the patient by teaching the robot intuitively through learning from demonstration. We then propose a learning control scheme based on dynamic movement primitives and iterative learning control, which takes the designed exercise trajectory as a demonstration (an input) together with the recorded forces in order to reproduce the exercise with the patient for a number of repetitions defined by the therapist. During the execution, our approach monitors the sensed forces and adapts the trajectory by adding the necessary offsets to the original trajectory to reduce its range without modifying the original trajectory and subsequently reducing the measured forces. After a predefined number of repetitions, the algorithm restores the range gradually, until the patient is able to perform the originally designed exercise. We validate the proposed framework with both real experiments and simulation using a Simulink model of the rehabilitation parallel robot that has been developed in our lab.
Acceso Abierto
Label-free optical biosensing using low-cost electrospun polymeric nanofibers
(MDPI Ag, 2020-12) Martínez Pérez, P.; Ponce Alcántara, S.; Murillo, Nieves; Pérez Márquez, Ana; Maudes, J.; Peraile, Inés; González López, L.; Gil García, M.; Lorenzo Lozano, P. ; García Rupérez, J.; Generalitat Valenciana (GVA); Ministerio de Economía y Competitividad (MINECO); Unidad de Excelencia Científica María de Maeztu Centro de Astrobiología del Instituto Nacional de Técnica Aeroespacial y CSIC, MDM-2017-0737
Polymeric nanofiber matrices are promising structures to develop biosensing devices due to their easy and affordable large-scale fabrication and their high surface-to-volume ratio. In this work, the suitability of a polyamide 6 nanofiber matrix for the development of a label-free and real-time Fabry–Pérot cavity-based optical biosensor was studied. For such aim, in-flow biofunctionalization of nanofibers with antibodies, bound through a protein A/G layer, and specific biodetection of 10 µg/mL bovine serum albumin (BSA) were carried out. Both processes were successfully monitored via reflectivity measurements in real-time without labels and their reproducibility was demonstrated when different polymeric nanofiber matrices from the same electrospinning batch were employed as transducers. These results demonstrate not only the suitability of correctly biofunctionalized polyamide 6 nanofiber matrices to be employed for real-time and label-free specific biodetection purposes, but also the potential of electrospinning technique to create affordable and easy-to-fabricate at large scale optical transducers with a reproducible performance.

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