Podcasts about taqman

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Background: Expression patterns of microRNAs in body fluids show potential to be used as noninvasive rapid and accurate biomarkers for various diseases.The study aimed to (i) identify patterns of microRNA signatures for diagnosis of tuberculosis (TB) and (ii) assess significance of a patient’s genetic background on signature composition and diagnostic performance. Patients and Methods: The study enrolled consented participants from Europe and Africa. Circulating miRNAs were measured and compared between patients belonging to the following categories; (i) active pulmonary tuberculosis (PTB), (ii) healthy individuals (H), (iii) active pulmonary TB co-infected with HIV (PTB/HIV), (iv) latent TB infection (LTBI) and (v) other pulmonary infection (OPI). As a first step, pooled sera of 10 participants from each category and region of enrolment were measured by TaqMan low-density arrays. Secondly, the identified significant miRNA signatures were applied to 56 individual sera aiming to discriminate between H and PTB patients. Next, the identified miRNA signatures were analysed for their diagnostic performances using multivariate logistic analysis, and Relevance Vector Machine (RVM). The diagnostic performance of both models was evaluated by a leave-one-out-cross-validation (LOOCV).

Classical and African swine fever belong to the most important contagious diseases of pigs worldwide and are notifiable to the World Organization for Animal Health (OIE). While outbreaks of classical swine fever (CSF) have a long and ongoing history in Europe, up to now African swine fever (ASF) was considered an exotic disease within EU Member States. However, very recently the disease has been introduced into the European wild boar and domestic pig population in Poland, Lithuania, Latvia, and Estland. Hence, both diseases pose a high risk to the whole European pig industry and wildlife. Regardless of very similar clinical pictures that are not discriminable without laboratory diagnosis, the causative agents differ greatly. Classical swine fever is caused by a small enveloped positive single-stranded RNA virus belonging to the genus Pestivirus within the Flaviviridae family. The causative agent of ASF is a complex DNA virus of the genus Asfivirus within the Asfarviridae family representing the only DNA arthropod-borne (ARBO) virus. Classical swine fever virus (CSFV) isolates of recent European outbreaks are characterized by their moderate virulence. The clinical picture can range from an almost inapparent infection to a lethal hemorrhagic fever like illness. High variability in disease course and outcome are a challenge for both disease surveillance and pathogenetic research. Although several studies aimed to analyze basic pathogenetic mechanisms, responsible factors have never been elucidated entirely. While on the host’s side, age and immune status are acknowleged parameters, the virulence of the isolate seems to be decisive on the agent’s side. Moreover, the influence of the genetic background of the host has been discussed. To define host responses linked to different disease courses and outcome, a first animal trial was conducted with a moderately virulent CSFV strain and different pig breeds including European wild boar. In a second trial, the impact of the age was revisited in combination with the assessment of tools for active swine fever surveillance. Dysregulation of immune responses, especially cytokine reactions, seems to play a crucial role in CSF pathogenesis. Up to now, there has been a serious lack of appropriate and reliable tools for cytokine gene expression analyses, especially in pigs. To overcome this shortcoming, a harmonized TaqMan-based RT-qPCR protocol for the detection of seven swine fever relevant cytokines was developed and fully validated. This assay is now available for future studies and could be implemented also for other swine diseases including ASF. Beyond the studies focusing on underlying mechanisms, diagnostic approaches for active and passive disease surveillance in wild boar have been targeted. Sample submission is usually the bottleneck of wildlife surveillance under field conditions, even in times of increased risk. Pragmatic approaches for sampling and transport could improve the compliance of hunters. In view of the fact that an introduction of ASF and CSF is usually accompanied by an increased mortality, animals found dead have to be sampled even if they are already in various stages of decay. To this means, a dry-/ semi-dry blood swab method was implemented enabling an easy handling under field and laboratory conditions. Moreover, a simple approach for sampling of live animals for active surveillance, i.e. a “rope-in-a-bait” method, was evaluated with respect to the frequently observed subclinical CSF forms in older animals.

Die gebräuchliche Therapie gegen Milzbrand besteht aus der Gabe von Antibiotika. Als Therapie der Wahl gilt hierbei das Fluorochinolon Ciprofloxacin. Resistenzen gegen dieses Antibiotikum wurden bei B. anthracis in vivo noch nicht, in vitro jedoch im Rahmen mehrerer Studien beschrieben. Es existieren herkömmliche Resistenztests, wie der Gradientendiffusions- oder der Mikrodilutionstest, welche bei einer Milzbranderkrankung genutzt werden können. Diese nehmen jedoch aufgrund der kulturellen Anzucht in einem Labor der Schutzstufe 3 vor der Durchführung des Tests ein bis zwei Tage Zeit in Anspruch. Um diese Zeitspanne zu verkürzen, wurden im Rahmen dieser Arbeit Schnelltests entwickelt. Diese basieren auf einer real-time-PCR Methode, mit welcher Ciprofloxacin-Resistenz verursachende Punktmutationen (= SNPs), nachgewiesen werden. Im ersten Abschnitt dieser Studie wurde der B. cereus Stamm ATCC10987 resistent gegen Ciprofloxacin generiert. Aufgrund der Dual-Use-Research-of-Concern-Problematik wurde dieser, wenig pathogene, aber genotypisch sehr nah mit B. anthracis verwandte, BSL-2-Organismus verwendet. Die Resistenzbildung erfolgte durch natürliche Selektion, indem der B. cereus Wildtyp mehrfach auf Ciprofloxacin-haltigen Agar-Platten, welche eine steigende Konzentration des Antibiotikums enthielten, angezüchtet wurde. Es folgte eine Sequenzierung der Quinolone Resistance Determinig Region (= QRDR), bestehend aus den Genen gyrA, gyrB, parC und parE, von neun B. cereus Mutanten, welche CIP-Resistenzen entwickelt hatten. Eine der Mutanten besaß einen SNP im Gen gyrA an Stelle 254 mit einer Mutation der Base Cytosin in ein Thymin. Solche SNPs stellen eine mögliche Ursache der Resistenz gegen Fluorochinolone dar. Acht der B. cereus Mutanten besaßen jedoch keine SNPs in der QRDR. Die Ursache für deren Resistenz wird in der erhöhten Funktion von Effluxpumpen vermutet. Im zweiten Teil der Studie wurden die Schnelltests entwickelt. Es wurden mehrere Protokolle für die beiden real-time-PCR Methoden TaqMan® und MeltMAMA (= Melt Analysis of Mismatch Amplification Mutation Assays) erstellt und getestet. Der Vergleich beider Methoden wertete den TaqMan® als die Methode der Wahl für die gesetzte Zielstellung. Daraufhin wurden für acht bekannte Ciprofloxacin-Resistenzen auslösende SNPs TaqMan®-Protokolle entwickelt. Im Abschluss wurden diese durch Versuche mit verschiedenen B. anthracis Stämmen, dem B. cereus ATCC10987 Wildtyp und seinen Mutanten, synthetisch hergestellten Templates, die als Mutationskontrollen genutzt wurden, sowie verschiedenen Bacillus Spezies hinsichtlich ihrer Sensitivität und Spezifität erprobt. Es wurden acht TaqMan® Protokolle erarbeitet, welche SNPs in der QRDR von B. anthracis nachweisen und somit eine schnelle Diagnose vieler Ciprofloxacin-resistenter Stämme gewährleisten. Der Einsatz dieser Schnelltests zusätzlich zu den herkömmlichen Empfindlichkeitstests gibt die Möglichkeit eine optimale Therapie von Milzbrandinfektionen in einem verkürzten Zeitraum zu gewährleisten.

Genetic variation in the IL28B gene has been strongly associated with treatment outcomes, spontaneous clearance and progression of the hepatitis C virus infection (HCV). The aim of the present study was to investigate the role of polymorphisms at this locus with progression and outcome of HCV infection in a Moroccan population. We analyzed a cohort of 438 individuals among them 232 patients with persistent HCV infection, of whom 115 patients had mild chronic hepatitis and 117 had advanced liver disease (cirrhosis and hepatocellular carcinoma), 68 individuals who had naturally cleared HCV and 138 healthy subjects. The IL28B SNPs rs12979860 and rs8099917 were genotyped using a TaqMan 5' allelic discrimination assay. The protective rs12979860-C and rs8099917-T alleles were more common in subjects with spontaneous clearance (77.9% vs 55.2%; p = 0.00001 and 95.6% vs 83.2%; p = 0.0025, respectively). Individuals with clearance were 4.69 (95% CI, 1.99-11.07) times more likely to have the C/C genotype for rs12979860 polymorphism (p = 0.0017) and 3.55 (95% CI, 0.19-66.89) times more likely to have the T/T genotype at rs8099917. Patients with advanced liver disease carried the rs12979860-T/T genotype more frequently than patients with mild chronic hepatitis C (OR = 1.89; 95% CI, 0.99-3.61; p = 0.0532) and this risk was even more pronounced when we compared them with healthy controls (OR = 4.27; 95% CI, 2.08-8.76; p = 0.0005). The rs8099917-G allele was also associated with advanced liver disease (OR = 2.34; 95% CI, 1.40-3.93; p = 0.0100). In the Moroccan population, polymorphisms near the IL28B gene play a role both in spontaneous clearance and progression of HCV infection.

Allergic asthma has a high prevalence and is characterized by airway inflammation, tissue remodeling and a decline in respiratory function. Although the pathogenesis is well known, the underlying mechanisms are still poorly understood. It is believed that a fine interplay exists between the exposure to environmental stimuli and relatively small changes in expression of several genes with inter-individual variation. As microRNAs (miRNAs) are known to be responsive to environmental exposures and show dysregulated levels in diseased states, their function as regulators of gene expression might be a missing link for the changes seen in asthma. In this project, changes in miRNA expression in a mouse model for allergic asthma were investigated and the interaction with possible target genes was analyzed. Female Balb/c mice were i. p. sensitized with ovalbumin (OVA) followed by aerosol challenge on two consecutive days. An asthmatic phenotype was confirmed by elevated total cell numbers due to a rise in inflammatory cells, as well as increased CCL17 levels in broncho-alveolar lavage (BAL). High titres of OVA-specific serum IgE were measured and lung histopathology revealed infiltration of inflammatory cells with eosinophilia. To study changes in miRNA expression, whole lung RNA was subjected to miRNA-microarray analysis (Exiqon). From 580 screened miRNAs, 319 were found to be expressed, of which 36 were differentially regulated in the allergic asthma group compared to healthy control mice. A second, TaqMan® chemistry based array was performed for validation. Based on the overlap between the two arrays in addition to fold changes and p-values (Exiqon), eight miRNAs were selected for single RT-qPCR measurement. Dysregulated expression of six miRNAs could be confirmed (miRNA-21, -142-3p, -144, -205, -208, -451). Due to relatively low fold changes and in order to monitor possible co-regulation, the top 100 differentially regulated miRNAs from the Exiqon array were included in an in situ target prediction. Applying a “full consensus” approach of five prediction algorithms, 961 putative target genes were identified. Based on the assumption, that target genes harboring multiple miRNA sites might be more relevant, 11 targets containing more than four miRNA binding sites were selected. From these, the transcription factor cAMP-responsive element-binding protein 1 (CREB1) was chosen for further analysis because of its previous association with asthmatic disease. Moreover, four miRNAs (miRNA-17, -22, -144, -181a) were predicted to bind at eight different sites, one of them being miRNA-144, a significantly up-regulated miRNA identified in the initial asthma profile. To experimentally test the functional interaction between CREB1 and the predicted miRNAs, a CREB1 3´-untranslated region (UTR) containing luciferase based reporter plasmid vector was constructed and co-transfected with precursor (pre-) miRNAs into human bronchial epithelial cells. Binding of all four miRNAs could be confirmed by measuring luciferase expression. Furthermore, three of four miRNAs, when transfected alone, were able to down-regulate endogenous CREB1 expression in vitro. In the lung tissue of asthma mice, CREB1 mRNA levels were significantly reduced compared to healthy controls, in contrast to two miRNAs, miRNA-17 and -144, which showed up-regulation. To gain further insight into expression patterns during sensitization and after challenge, expression of CREB1, the two validated binding partners miRNA-17, and -144, as well as the two miRNAs (miRNA-21, -451) with most significant p-values and high fold changes from the Exiqon array were analyzed. Clear expression changes happened after OVA aerosol challenge with CREB1 levels being steadily decreased, whereas all tested miRNAs showed elevated levels at 24 h post challenge, which further intensified after 120 h. This increase resembles measurements of inflammatory cell counts in BAL pointing at a possible origin within this population. In order to test whether findings can be translated into the human situation, miRNA changes in whole blood samples of mice were compared to miRNA patterns in peripheral blood of asthmatic children. In contrast to measurements in lung tissue, all four miRNAs showed markedly decreased expression in murine blood. In human samples this reduction was mirrored and significant for miRNA-144 and -451.

Aim: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. Results: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 mu g/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. Conclusions: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.