Real-time PCR optimization to identify Mycobacterium tuberculosis complex strains in clinical samples.

Resumen del artículo publicado en FEBS Journal,http://dx.doi.org/10.1111/febs.12919During recent years several molecular techniques have become available for Mycobacterium tuberculosis complex (MTC) detection,both for clinical samples and for isolates. One of the techniques more widely used is real time PCR in combination with nucleic acid amplification protocols. There are numerous studies based on PCR for the diagnosis of tuberculosis although the different protocols and primers used in the laboratory, together with the variability in the diagnostic performance of the methods tested, require that a comparative study be performed. Furthermore,the fact that the detection from clinical samples requires using highly sensitive targets suggests that this type of study should include multicopy targets to compare their efficiency with respect to the single copy. Our aim was to identify the members of the MTC using real-time PCR assays based on SYBR Green,among a large panel of isolated bacterial strains and clinical samples.We chose three targets (IS6110, senx3-regx3 and cfp32) and the optimal values for each PCR assay were empirically defined by testing in triplicate different concentrations of MgCl2 and primer sets and different annealing temperatures. These conditions were determined based on the specific amplification reactions that showed a lower Ct value, higher fluorescence and absence of non-specific PCR products. The analytical sensitivity was evaluated by ten-fold serial dilutions of DNA from MTC and the specificity was tested by 62 different microorganisms, including bacteria related with the MTC. The diagnostic yield was evaluated in 66 specimens from patients with suspected tuberculosis;30 had tuberculosis and 36 (control group) had different diseases.Under the conditions that resulted in optimization, standard curves showed that senx3-regx3 assay was the most efficient, followed by IS6110 and cfp32. However, the detection of bacterial DNA was faster with the repetitive element IS6110, with Ct values of up to 3 and 9 cycles of difference with respect to senx3-regx3 and cfp32. The analytical specificity, done only with the senx3-regx3 and IS6110 targets, was in the order of 100 and 93.5%, since IS6110 amplified various non-tuberculous micobacteria.For all the clinical samples studied, the sensitivity of both assays was identical (93.3%) but the specificity of senx3-regx3(100%) was higher than that of IS6110 (94.7%). In conclusion,real time PCR assay-SYBR Green based on the targets senx3-regx3 is highly reproducible and more sensitive and specific than the assays based on IS6110 or cfp32. The protocol developed in this study provides an appropriate and rapid tool to identify the strains of MTC in different clinical isolates and specimens.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

La influencia de la tecnología en los procesos de producción musical: análisis de la grabación sonora y sus aplicaciones en el ámbito docente

Memoria de las actividades del proyecto de Innova-Docencia nº256, coordinado por el Dr. Marco Antonio Juan de Dios Cuartas, profesor del Departamento de Musicología de la Universidad Complutense de Madrid

El laboratorio sonoro como herramienta docente para el análisis de la creación e interpretación musical a través de las grabaciones

Depto. de MusicologíaFac. de Geografía e HistoriaFALSEsubmitte

Strategy for Optimizing DNA Amplification in a Peripheral Blood PCR Assay Used for Diagnosis of Human Brucellosis

We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 μg, thereby avoiding false-negative results

Procedimiento de detección de secuencias de ADN específicas de Brucella spp mediante reacción en cadena de la polimerasa (PCR) acoplada a un enzimoinmunoensayo (ELISA)

Número de publicación: 2 220 180 Número de solicitud: 200201381Procedimiento de detección de secuencias de ADN específicas de Brucella spp mediante reacción en cadena de la polimerasa (PCR) acoplada a un enzimoinmunoensayo (ELISA). Procedimiento de detección de secuencias de ADN específicas de Brucella spp. mediante reacción en cadena de la polimerasa (PCR) acoplada a un enzimoinmunoensayo(ELISA). Después de una amplificación selectiva de una secuencia de 223 pb de Brucella utilizando los oligonucleótidos B4 y B5, los productos amplificados resultantes marcados con digoxigenina (DIG) son hibridados con una sonda de captura biotinilada. Posteriormente, estos híbridos son capturados en placas recubiertas con estreptavidina y detectados usando un conjugado anti-DIG Fab/ peroxidasa. La técnica propuesta se ha mostrado mucho mas sensible que la PCR convencional y que los métodos bacteriológicos y mas específica que los métodos serológicos habituales, permitiendo su utilización para la puesta en práctica de un procedimiento fácil y rápido de diagnóstico molecular de la infección por Brucella spp. en muestras de sangre humana y en otras muestras clínicas, la monitorización de la respuesta al tratamiento y la detección precoz de las recidivas evitando el riesgo de manipulación del germen.Universidad de Granad

Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis.

Journal Article; Research Support, Non-U.S. Gov't;Real-time PCR is a widely used tool for the diagnosis of many infectious diseases. However, little information exists about the influences of the different factors involved in PCR on the amplification efficiency. The aim of this study was to analyze the effect of boiling as the DNA preparation method on the efficiency of the amplification process of real-time PCR for the diagnosis of human brucellosis with serum samples. Serum samples from 10 brucellosis patients were analyzed by a SYBR green I LightCycler-based real-time PCR and by using boiling to obtain the DNA. DNA prepared by boiling lysis of the bacteria isolated from serum did not prevent the presence of inhibitors, such as immunoglobulin G (IgG), which were extracted with the template DNA. To identify and confirm the presence of IgG, serum was precipitated to separate and concentrate the IgG and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The use of serum volumes above 0.6 ml completely inhibited the amplification process. The inhibitory effect of IgG in serum samples was not concentration dependent, and it could be eliminated by diluting the samples 1/10 and 1/20 in water. Despite the lack of the complete elimination of the IgG from the template DNA, boiling does not require any special equipment and it provides a rapid, reproducible, and cost-effective method for the preparation of DNA from serum samples for the diagnosis of brucellosis.This study was supported by Red Tematica para la Investigacion en Brucelosi, Instituto de Salud Carlos III (ISCIII), grant PI 05/1733; Servicio Andaluz de Salud (SAS) grant 152/04; and Consejeria de Innovación Ciencia y Empresa (GI 2005, CTS-276).Ye

Application of New Conformal Cooling Layouts to the Green Injection Molding of Complex Slender Polymeric Parts with High Dimensional Specifications

Eliminating warpage in injection molded polymeric parts is one of the most important problems in the injection molding industry today. This situation is critical in geometries that are particularly susceptible to warping due to their geometric features, and this occurs with topologies of great length and slenderness with high changes in thickness. These features are, in these special geometries, impossible to manufacture with traditional technologies to meet the dimensional and sustainable requirements of the industry. This paper presents an innovative green conformal cooling system that is specifically designed for parts with slender geometric shapes that are highly susceptible to warping. Additionally, the work presented by the authors investigates the importance of using highly conductive inserts made of steel alloys in combination with the use of additively manufactured conformal channels for reducing influential parameters, such as warpage, cooling time, and residual stresses in the complex manufacturing of long and slender parts. The results of this real industrial case study indicated that the use of conformal cooling layouts decreased the cycle time by 175.1 s—66% below the current cooling time; the temperature gradient by 78.5%—specifically, 18.16 °C; the residual stress by 39.78 MPa—or 81.88%; and the warpage by 6.9 mm—or 90.5%. In this way, it was possible to achieve a final warping in the complex geometry studied of 0.72 mm, which was under the maximum value required at the industrial level of 1 mm. The resulting values obtained by the researchers present a turning point from which the manufacturing and sustainability in the injection molding of said plastic geometries is possible, and they take into account that the geometric manufacturing features analyzed will present a great demand in the coming years in the auto parts manufacturing industry